Peptides whose uptake in cells is controllable

ABSTRACT

Disclosed herein, in certain embodiments, is a selective transport molecule with increased in vivo circulation. In some embodiments, a selective transport molecule disclosed herein has the formula (A-X-B-C)n-M, wherein C is a cargo moiety; A is a peptide with a sequence comprising 5 to 9 consecutive acidic amino acids, wherein the amino acids are selected from: aspartates and glutatmates; B is a peptide with a sequence comprising 5 to 20 consecutive basic amino acids; X is a linker; and M is a macromolecular carrier.

CROSS-REFERENCE

This application is the National Stage of International Application No.PCT/US2010/042188, filed Jul. 15, 2010, which claims the benefit of U.S.Provisional Application No. 61/225,872, filed Jul. 15, 2009, whichapplications are incorporated herein by reference in its entirety.

STATEMENT AS TO FEDERALLY SPONSORED RESEARCH

This invention was made with governmental support under grant no.W81XWH-05-1-0183 awarded by the US Army Department of Defense, and grantno. NIBIB-K08 EB008122 awarded by the NIH. The government has certainrights in the invention.

BACKGROUND OF THE INVENTION

Cell membranes delimit the outer boundaries of cells, and regulatetransport into and out of the cell interior. Made primarily of lipidsand proteins, they provide a hydrophilic surface enclosing a hydrophobicinterior across which materials must pass before entering a cell.Although many small, lipophilic compounds are able to cross cellmembranes passively, most compounds, particles and materials must relyon active mechanisms in order to gain entry into a living cell.

SUMMARY OF THE INVENTION

Disclosed herein, in certain embodiments, is a molecule for imagingthrombin activity in a subject, wherein the molecule has the formula:(A-X-B-C)_(n)-M, wherein

-   -   C is at least one imaging agent;    -   A is a peptide with a sequence comprising 5 to 9 consecutive        acidic amino acids, wherein the amino acids are selected from:        aspartates and glutamates;    -   B is a peptide with a sequence comprising 5 to 20 consecutive        basic amino acids;    -   X is a thrombin cleavable linker;    -   M is a macromolecular carrier; and    -   n is an integer between 1 and 20; and    -   wherein M is bound to A or B.        In some embodiments, A has a sequence comprising 5 to 9        consecutive glutamates. In some embodiments, B has a sequence        comprising 5 to 12 consecutive arginines. In some embodiments, B        has a sequence comprising 9 consecutive arginines. In some        embodiments, (a) A has a sequence comprising 8 to 9 consecutive        glutamates and (b) B has a sequence comprising 9 consecutive        arginines. In some embodiments, A and B comprise D-amino acids.        In some embodiments, X comprises a peptide linkage. In some        embodiments, X comprises 6-aminohexanoyl,        5-amino-3-oxapentanoyl, or a combination thereof. In some        embodiments, X comprises a disulfide linkage. In some        embodiments, X is about 6 to about 30 atoms in length. In some        embodiments, the X is selected from: DPRSFL (SEQ ID NO: 1), or        PPRSFL (SEQ ID NO: 2). In some embodiments, M is a        macromolecular carrier selected from: a dendrimer, dextran, a        PEG polymer, albumin, or lipid-coated perfluorocarbon droplet.        In some embodiments, M is a PEG polymer. In some embodiments,        the imaging agent is selected from: a fluorescent moiety, a        luminescent moiety, a phosphorescent moiety, a        fluorescence-quenching moiety, a radioactive moiety, a        radiopaque moiety, a paramagnetic moiety, a contrast agent,        ultrasound scatterer, or a combination thereof. In some        embodiments, the imaging agent is selected from an        indocarbocyanine dye. In some embodiments, the imaging agent is        selected from Cy5, Cy5.5, Cy7, Alexa 647, IRDYE 800CW, or a        combination thereof. In some embodiments, the imaging agent is        an MRI contrast agent. In some embodiments, the imaging agent is        Gd complex of        [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.

Disclosed herein, in certain embodiments, is a method of imagingthrombin activity in a subject, comprising imaging thrombin activityafter the subject has been administered a molecule of the structure(A-X-B-C)_(n)-M, wherein

-   -   C is at least one imaging agent;    -   A is a peptide with a sequence comprising 5 to 9 consecutive        acidic amino acids, wherein the amino acids are selected from:        aspartates and glutamates;    -   B is a peptide with a sequence comprising 5 to 20 consecutive        basic amino acids;    -   X is a thrombin cleavable linker;    -   M is a macromolecular carrier;    -   n is an integer between 1 and 20; and    -   wherein M is bound to A or B.        In some embodiments, A has a sequence comprising 5 to 9        consecutive glutamates. In some embodiments, B has a sequence        comprising 5 to 12 consecutive arginines. In some embodiments, B        has a sequence comprising 9 consecutive arginines. In some        embodiments, (a) A has a sequence comprising 8 to 9 consecutive        glutamates and (b) B has a sequence comprising 9 consecutive        arginines. In some embodiments, A and B comprise D-amino acids.        In some embodiments, X comprises a peptide linkage. In some        embodiments, X comprises 6-aminohexanoyl,        5-amino-3-oxapentanoyl, or a combination thereof. In some        embodiments, X comprises a disulfide linkage. In some        embodiments, X is about 6 to about 30 atoms in length. In some        embodiments, the X is selected from: DPRSFL (SEQ ID NO: 1), or        PPRSFL (SEQ ID NO: 2). In some embodiments, M is a        macromolecular carrier selected from: a dendrimer, dextran, a        PEG polymer, albumin, or lipid-coated perfluorocarbon droplet.        In some embodiments, M is a PEG polymer. In some embodiments,        the imaging agent is selected from: a fluorescent moiety, a        luminescent moiety, a phosphorescent moiety, a        fluorescence-quenching moiety, a radioactive moiety, a        radiopaque moiety, a paramagnetic moiety, a contrast agent,        ultrasound scatterer, or a combination thereof. In some        embodiments, the imaging agent is selected from an        indocarbocyanine dye. In some embodiments, the imaging agent is        selected from Cy5, Cy5.5, Cy7, Alexa 647, IRDYE 800CW, or a        combination thereof. In some embodiments, the imaging agent is        an MRI contrast agent. In some embodiments, the imaging agent is        Gd complex of        [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.

Disclosed herein, in certain embodiments, is a use of a molecule to ofstructure (A-X-B-C)_(n)-M for visualizing thrombin activity in a subjectin need thereof, wherein

-   -   C is at least one imaging agent;    -   A is a peptide with a sequence comprising 5 to 9 consecutive        acidic amino acids, wherein the amino acids are selected from:        aspartates and glutamates;    -   B is a peptide with a sequence comprising 5 to 20 consecutive        basic amino acids;    -   X is a thrombin cleavable linker;    -   M is a macromolecular carrier; and    -   n is an integer between 1 and 20; and    -   wherein M is bound to A or B.        In some embodiments, A has a sequence comprising 5 to 9        consecutive glutamates. In some embodiments, B has a sequence        comprising 5 to 12 consecutive arginines. In some embodiments, B        has a sequence comprising 9 consecutive arginines. In some        embodiments, (a) A has a sequence comprising 8 to 9 consecutive        glutamates and (b) B has a sequence comprising 9 consecutive        arginines. In some embodiments, A and B comprise D-amino acids.        In some embodiments, X comprises a peptide linkage. In some        embodiments, X comprises 6-aminohexanoyl,        5-amino-3-oxapentanoyl, or a combination thereof. In some        embodiments, X comprises a disulfide linkage. In some        embodiments, X is about 6 to about 30 atoms in length. In some        embodiments, the X is selected from: DPRSFL (SEQ ID NO: 1), or        PPRSFL (SEQ ID NO: 2). In some embodiments, M is a        macromolecular carrier selected from: a dendrimer, dextran, a        PEG polymer, albumin, or lipid-coated perfluorocarbon droplet.        In some embodiments, M is a PEG polymer. In some embodiments,        the imaging agent is selected from: a fluorescent moiety, a        luminescent moiety, a phosphorescent moiety, a        fluorescence-quenching moiety, a radioactive moiety, a        radiopaque moiety, a paramagnetic moiety, a contrast agent,        ultrasound scatterer, or a combination thereof. In some        embodiments, the imaging agent is selected from an        indocarbocyanine dye. In some embodiments, the imaging agent is        selected from Cy5, Cy5.5, Cy7, Alexa 647, IRDYE 800CW, or a        combination thereof. In some embodiments, the imaging agent is        an MRI contrast agent. In some embodiments, the imaging agent is        Gd complex of        [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.

Disclosed herein, in certain embodiments, is a method of characterizingthe risk associated with an atherosclerotic plaque in a subject,comprising imaging thrombin activity in the atherosclerotic plaque afterthe subject has been administered a molecule of the structure(A-X-B-C)_(n)-M,

-   -   C is at least one imaging agent;    -   A is a peptide with a sequence comprising 5 to 9 consecutive        acidic amino acids, wherein the amino acids are selected from:        aspartates and glutamates;    -   B is a peptide with a sequence comprising 5 to 20 consecutive        basic amino acids;    -   X is a thrombin cleavable linker;    -   M is a macromolecular carrier;    -   n is an integer between 1 and 20; and    -   wherein M is bound to A or B; and    -   wherein the risk is proportional to the imaging agent signal        intensity.        In some embodiments, A has a sequence comprising 5 to 9        consecutive glutamates. In some embodiments, B has a sequence        comprising 5 to 12 consecutive arginines. In some embodiments, B        has a sequence comprising 9 consecutive arginines. In some        embodiments, (a) A has a sequence comprising 8 to 9 consecutive        glutamates and (b) B has a sequence comprising 9 consecutive        arginines. In some embodiments, A and B comprise D-amino acids.        In some embodiments, X comprises a peptide linkage. In some        embodiments, X comprises 6-aminohexanoyl,        5-amino-3-oxapentanoyl, or a combination thereof. In some        embodiments, X comprises a disulfide linkage. In some        embodiments, X is about 6 to about 30 atoms in length. In some        embodiments, the X is selected from: DPRSFL (SEQ ID NO: 1), or        PPRSFL (SEQ ID NO: 2). In some embodiments, M is a        macromolecular carrier selected from: a dendrimer, dextran, a        PEG polymer, albumin, or lipid-coated perfluorocarbon droplet.        In some embodiments, M is a PEG polymer. In some embodiments,        the imaging agent is selected from: a fluorescent moiety, a        luminescent moiety, a phosphorescent moiety, a        fluorescence-quenching moiety, a radioactive moiety, a        radiopaque moiety, a paramagnetic moiety, a contrast agent,        ultrasound scatterer, or a combination thereof. In some        embodiments, the imaging agent is selected from an        indocarbocyanine dye. In some embodiments, the imaging agent is        selected from Cy5, Cy5.5, Cy7, Alexa 647, IRDYE 800CW, or a        combination thereof. In some embodiments, the imaging agent is        an MRI contrast agent. In some embodiments, the imaging agent is        Gd complex of        [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.

Disclosed herein, in certain embodiments, is a use of a molecule of thestructure (A-X-B-C)_(n)-M to characterize the risk associated with anatherosclerotic plaque in a subject, wherein

-   -   C is at least one imaging agent;    -   B is a peptide with a sequence comprising 5 to 20 consecutive        basic amino acids;    -   X is a thrombin cleavable linker;    -   M is a macromolecular carrier;    -   n is an integer between 1 and 20; and    -   wherein M is bound to A or B; and    -   wherein the risk is proportional to the imaging agent signal        intensity.        In some embodiments, A has a sequence comprising 5 to 9        consecutive glutamates. In some embodiments, B has a sequence        comprising 5 to 12 consecutive arginines. In some embodiments, B        has a sequence comprising 9 consecutive arginines. In some        embodiments, (a) A has a sequence comprising 8 to 9 consecutive        glutamates and (b) B has a sequence comprising 9 consecutive        arginines. In some embodiments, A and B comprise D-amino acids.        In some embodiments, X comprises a peptide linkage. In some        embodiments, X comprises 6-aminohexanoyl,        5-amino-3-oxapentanoyl, or a combination thereof. In some        embodiments, X comprises a disulfide linkage. In some        embodiments, X is about 6 to about 30 atoms in length. In some        embodiments, the X is selected from: DPRSFL (SEQ ID NO: 1), or        PPRSFL (SEQ ID NO: 2). In some embodiments, M is a        macromolecular carrier selected from: a dendrimer, dextran, a        PEG polymer, albumin, or lipid-coated perfluorocarbon droplet.        In some embodiments, M is a PEG polymer. In some embodiments,        the imaging agent is selected from: a fluorescent moiety, a        luminescent moiety, a phosphorescent moiety, a        fluorescence-quenching moiety, a radioactive moiety, a        radiopaque moiety, a paramagnetic moiety, a contrast agent,        ultrasound scatterer, or a combination thereof. In some        embodiments, the imaging agent is selected from an        indocarbocyanine dye. In some embodiments, the imaging agent is        selected from Cy5, Cy5.5, Cy7, Alexa 647, IRDYE 800CW, or a        combination thereof. In some embodiments, the imaging agent is        an MRI contrast agent. In some embodiments, the imaging agent is        Gd complex of        [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.

Disclosed herein, in certain embodiments, is a method of treating acardiovascular disorder characterized by an increase in thrombinactivity as compared to a subject without the cardiovascular disorder,comprising administering to the subject a molecule of the structure(A-X-B-C)_(n)-M,

-   -   C is at least one therapeutic agent;    -   A is a peptide with a sequence comprising 5 to 9 consecutive        acidic amino acids, wherein the amino acids are selected from:        aspartates and glutamates;    -   B is a peptide with a sequence comprising 5 to 20 consecutive        basic amino acids;    -   X is a thrombin cleavable linker;    -   M is a macromolecular carrier;    -   n is an integer between 1 and 20; and    -   wherein M is bound to A or B.        In some embodiments, A has a sequence comprising 5 to 9        consecutive glutamates. In some embodiments, B has a sequence        comprising 5 to 12 consecutive arginines. In some embodiments, B        has a sequence comprising 9 consecutive arginines. In some        embodiments, (a) A has a sequence comprising 8 to 9 consecutive        glutamates and (b) B has a sequence comprising 9 consecutive        arginines. In some embodiments, A and B comprise D-amino acids.        In some embodiments, X comprises a peptide linkage. In some        embodiments, X comprises 6-aminohexanoyl,        5-amino-3-oxapentanoyl, or a combination thereof. In some        embodiments, X comprises a disulfide linkage. In some        embodiments, X is about 6 to about 30 atoms in length. In some        embodiments, the X is selected from: DPRSFL (SEQ ID NO: 1), or        PPRSFL (SEQ ID NO: 2). In some embodiments, M is a        macromolecular carrier selected from: a dendrimer, dextran, a        PEG polymer, albumin, or lipid-coated perfluorocarbon droplet.        In some embodiments, M is a PEG polymer. In some embodiments,        the therapeutic agent is an HDL-raising therapy, a Glycoprotein        (GP) IIb/IIIa receptor antagonist, a P2Y12 receptor antagonist,        a Lp-PLA2-inhibitor, a leukotriene inhibitor, a MIF antagonist,        or a combination thereof. In some embodiments, the therapeutic        agent is: a niacin, a fibrate, a statin, an Apo-A1 mimetic        peptide, an apoA-I transciptional up-regulator, an ACAT        inhibitor, a CETP modulator, or a combination thereof. In some        embodiments, the therapeutic agent is: atorvastatin;        cerivastatin; fluvastatin; lovastatin; mevastatin; pitavastatin;        pravastatin; rosuvastatin; simvastatin; simvastatin and        ezetimibe; lovastatin and niacin, extended-release; atorvastatin        and amlodipine besylate; simvastatin and niacin,        extended-release; bezafibrate; ciprofibrate; clofibrate;        gemfibrozil; fenofibrate; DF4        (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (SEQ ID NO: 3));        DFS; RVX-208 (Resverlogix); avasimibe; pactimibe sulfate        (CS-505); CI-1011 (2,6-diisopropylphenyl [(2,        4,6-triisopropylphenylacetyl]sulfamate); CI-976        (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); VULM1457        (1-(2,6-diisopropyl-phenyl)-3-[4-(4′-nitrophenylthio)phenyl]        urea); CI-976        (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); E-5324        (n-butyl-N′-(2-(3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy)-6-methylphenyl)urea);        HL-004 (N-(2,6-diisopropylphenyl) tetradecylthioacetamide);        KY-455        (N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide);        FY-087        (N-[2-[N′-pentyl-(6,6-dimethyl-2,4-heptadiynyl)amino]ethyl]-(2-methyl-1-naphthyl-thio)acetamide);        MCC-147 (Mitsubishi Pharma); F 12511        ((S)-2′,3′,5′-trimethyl-4′-hydroxy-alpha-dodecylthioacetanilide);        SMP-500 (Sumitomo Pharmaceuticals); CL 277082        (2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropylphenyl]methyl]-N-(hepthyl]urea);        F-1394        ((1s,2s)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]aminocyclohexane-1-yl        3-[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate);        CP-113818        (N-(2,4-bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic        acid amide); YM-750; torcetrapib; anacetrapid; JTT-705 (Japan        Tobacco/Roche); abciximab; eptifibatide; tirofiban; roxifiban;        variabilin; XV 459        (N(3)-(2-(3-(4-formamidinophenyl)isoxazolin-5-yl)acetyl)-N(2)-(1-butyloxycarbonyl)-2,3-diaminopropionate);        SR 121566A        (3-[N-{4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl}-N-(1-carboxymethylpiperid-4-yl)        aminol propionic acid, trihydrochloride); FK419        ((S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl) propionyl]        piperidin-3-ylcarbonyl] amino] propionic acid trihydrate);        clopidogrel; prasugrel; cangrelor; AZD6140 (AstraZeneca); MRS        2395 (2,2-Dimethyl-propionic acid        3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl        ester); BX 667 (Berlex Biosciences); BX 048 (Berlex        Biosciences); darapladib (SB 480848); SB-435495        (GlaxoSmithKline); SB-222657 (GlaxoSmithKline); SB-253514        (GlaxoSmithKline); A-81834        (3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde        oxime-O-2-acetic acid; AME103 (Amira); AME803 (Amira);        atreleuton; BAY-x-1005        ((R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic        acid); CJ-13610        (4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylic        acid amide); DG-031 (DeCode); DG-051 (DeCode); MK886        (1-[(4-chlorophenylmethyl]3-[(1,1-dimethylethyl)thio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoic        acid, sodium salt); MK591        (3-(1-4[(4-chlorophenyl)methyl]-3-[(t-butylthio)-5-((2-quinoly)methoxy)-1H-indole-2]-,        dimehtylpropanoic acid); RP64966        ([4-[5-(3-Phenyl-propyl)thiophen-2-yl]butoxy] acetic acid);        SA6541        ((R)-S-[[4-(dimethylamino)phenyl]methyl]-N-(3-mercapto-2methyl-1-oxopropyl-L-cycteine);        SC-56938 (ethyl-1-[2-[4-(phenylmethyl)phenoxy]        ethyl]-4-piperidine-carboxylate); VIA-2291 (Via        Pharmaceuticals); WY-47,288        (2-[(1-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138        (6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone);        or combinations thereof. In some embodiments, C further        comprises at least one imaging agent. In some embodiments, C        further comprises: a fluorescent moiety, a luminescent moiety, a        phosphorescent moiety, a fluorescence-quenching moiety, a        radioactive moiety, a radiopaque moiety, a paramagnetic moiety,        a contrast agent, ultrasound scatterer, or a combination        thereof. In some embodiments, C further comprises an        indocarbocyanine dye. In some embodiments, C further comprises:        Cy5, Cy5.5, Cy7, Alexa 647, IRDYE 800CW, or a combination        thereof. In some embodiments, C further comprises an MRI        contrast agent. In some embodiments, C further comprises Gd        complex of        [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.

Disclosed herein, in certain embodiments, is a use of a molecule of thestructure (A-X-B-C)_(n)-M to treat a cardiovascular disordercharacterized by an increase in thrombin activity as compared to asubject without the cardiovascular disorder, wherein

-   -   C is at least one therapeutic agent;    -   A is a peptide with a sequence comprising 5 to 9 consecutive        acidic amino acids, wherein the amino acids are selected from:        aspartates and glutamates;    -   B is a peptide with a sequence comprising 5 to 20 consecutive        basic amino acids;    -   X is a thrombin cleavable linker;    -   M is a macromolecular carrier;    -   n is an integer between 1 and 20; and    -   wherein M is bound to A or B.        In some embodiments, A has a sequence comprising 5 to 9        consecutive glutamates. In some embodiments, B has a sequence        comprising 5 to 12 consecutive arginines. In some embodiments, B        has a sequence comprising 9 consecutive arginines. In some        embodiments, (a) A has a sequence comprising 8 to 9 consecutive        glutamates and (b) B has a sequence comprising 9 consecutive        arginines. In some embodiments, A and B comprise D-amino acids.        In some embodiments, X comprises a peptide linkage. In some        embodiments, X comprises 6-aminohexanoyl,        5-amino-3-oxapentanoyl, or a combination thereof. In some        embodiments, X comprises a disulfide linkage. In some        embodiments, X is about 6 to about 30 atoms in length. In some        embodiments, the X is selected from: DPRSFL (SEQ ID NO: 1), or        PPRSFL (SEQ ID NO: 2). In some embodiments, M is a        macromolecular carrier selected from: a dendrimer, dextran, a        PEG polymer, albumin, or lipid-coated perfluorocarbon droplet.        In some embodiments, M is a PEG polymer. In some embodiments,        the therapeutic agent is an HDL-raising therapy, a Glycoprotein        (GP) Hb/IIIa receptor antagonist, a P2Y12 receptor antagonist, a        Lp-PLA2-inhibitor, a leukotriene inhibitor, a MIF antagonist, or        a combination thereof. In some embodiments, the therapeutic        agent is: a niacin, a fibrate, a statin, an Apo-A1 mimetic        peptide, an apoA-I transciptional up-regulator, an ACAT        inhibitor, a CETP modulator, or a combination thereof. In some        embodiments, the therapeutic agent is: atorvastatin;        cerivastatin; fluvastatin; lovastatin; mevastatin; pitavastatin;        pravastatin; rosuvastatin; simvastatin; simvastatin and        ezetimibe; lovastatin and niacin, extended-release; atorvastatin        and amlodipine besylate; simvastatin and niacin,        extended-release; bezafibrate; ciprofibrate; clofibrate;        gemfibrozil; fenofibrate; DF4        (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (SEQ ID NO: 3));        DFS; RVX-208 (Resverlogix); avasimibe; pactimibe sulfate        (CS-505); CI-1011 (2,6-diisopropylphenyl [(2,        4,6-triisopropylphenyl)acetyl]sulfamate); CI-976        (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); VULM1457        (1-(2,6-diisopropyl-phenyl)-3-[4-(4′-nitrophenylthio)phenyl]        urea); CI-976        (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); E-5324        (n-butyl-N′-(2-(3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy)-6-methylphenyl)urea);        HL-004 (N-(2,6-diisopropylphenyl) tetradecylthioacetamide);        KY-455        (N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide);        FY-087        (N-[2-[N′-pentyl-(6,6-dimethyl-2,4-heptadiynylamino]ethyl]-(2-methyl-1-naphthyl-thio)acetamide);        MCC-147 (Mitsubishi Pharma); F 12511        ((S)-2′,3′,5′-trimethyl-4′-hydroxy-alpha-dodecylthioacetanilide);        SMP-500 (Sumitomo Pharmaceuticals); CL 277082        (2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropyl)phenyl]methyl]-N-(hepthyl)urea);        F-1394 ((1        s,2s)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]aminocyclohexane-1-yl        3-[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate);        CP-113818        (N-(2,4-bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic        acid amide); YM-750; torcetrapib; anacetrapid; JTT-705 (Japan        Tobacco/Roche); abciximab; eptifibatide; tirofiban; roxifiban;        variabilin; XV 459        (N(3)-(2-(3-(4-formamidinophenyl)isoxazolin-5-yl)acetyl)-N(2)-(1-butyloxycarbonyl)-2,3-diaminopropionate);        SR 121566A        (3-[N-{-4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl}-N-(1-carboxymethylpiperid-4-yl)        aminol propionic acid, trihydrochloride); FK419        ((S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl) propionyl]        piperidin-3-ylcarbonyl] amino] propionic acid trihydrate);        clopidogrel; prasugrel; cangrelor; AZD6140 (AstraZeneca); MRS        2395 (2,2-Dimethyl-propionic acid        3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propyl        ester); BX 667 (Berlex Biosciences); BX 048 (Berlex        Biosciences); darapladib (SB 480848); SB-435495        (GlaxoSmithKline); SB-222657 (GlaxoSmithKline); SB-253514        (GlaxoSmithKline); A-81834        (3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehyde        oxime-O-2-acetic acid; AME103 (Amira); AME803 (Amira);        atreleuton; BAY-x-1005        ((R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic        acid); CJ-13610        (4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylic        acid amide); DG-031 (DeCode); DG-051 (DeCode); MK886        (1-[(4-chlorophenyl]methyl]3-[(1,1-dimethylethyl)thio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoic        acid, sodium salt); MK591        (3-(1-4[(4-chlorophenyl)methyl]-3-[(t-butylthio)-5-((2-quinoly)methoxy)-1H-indole-2]-,        dimehtylpropanoic acid); RP64966        ([4-[5-(3-Phenyl-propyl)thiophen-2-yl]butoxy] acetic acid);        SA6541        ((R)-S-[[4-(dimethylamino)phenyl]methyl]-N-(3-mercapto-2methyl-1-oxopropyl-L-cycteine);        SC-56938 (ethyl-l-[2-[4-(phenylmethyl)phenoxy]        ethyl]-4-piperidine-carboxylate); VIA-2291 (Via        Pharmaceuticals); WY-47,288        (2-[(1-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138        (6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone);        or combinations thereof. In some embodiments, C further        comprises at least one imaging agent. In some embodiments, C        further comprises: a fluorescent moiety, a luminescent moiety, a        phosphorescent moiety, a fluorescence-quenching moiety, a        radioactive moiety, a radiopaque moiety, a paramagnetic moiety,        a contrast agent, ultrasound scatterer, or a combination        thereof. In some embodiments, C further comprises an        idocarbocyanine dye. In some embodiments, C further comprises:        Cy5, Cy5.5, Cy7, Alexa 647, IRDYE 800CW, or a combination        thereof. In some embodiments, C further comprises an MRI        contrast agent. In some embodiments, C further comprises Gd        complex of        [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]        acetyl.

Disclosed herein, in certain embodiments, is an image of thrombinactivity, wherein the image is generated by imaging thrombin activityafter the subject has been administered a molecule of the structure(A-X-B-C)_(n)-M, wherein

-   -   C is at least one imaging agent;    -   A is a peptide with a sequence comprising 5 to 9 consecutive        acidic amino acids, wherein the amino acids are selected from:        aspartates and glutamates;    -   B is a peptide with a sequence comprising 5 to 20 consecutive        basic amino acids;    -   X is a thrombin cleavable linker;    -   M is a macromolecular carrier;    -   n is an integer between 1 and 20; and    -   wherein M is bound to A or B.        In some embodiments, the image is stored in analogue form or        digital form. In some embodiments, the image is stored as a        photograph. In some embodiments, the image is stored on a        computer readable medium. In some embodiments, A has a sequence        comprising 5 to 9 consecutive glutamates. In some embodiments, B        has a sequence comprising 5 to 12 consecutive arginines. In some        embodiments, B has a sequence comprising 9 consecutive        arginines. In some embodiments, (a) A has a sequence comprising        8 to 9 consecutive glutamates and (b) B has a sequence        comprising 9 consecutive arginines. In some embodiments, A and B        comprise D-amino acids. In some embodiments, X comprises a        peptide linkage. In some embodiments, X comprises        6-aminohexanoyl, 5-amino-3-oxapentanoyl, or a combination        thereof. In some embodiments, X comprises a disulfide linkage.        In some embodiments, X is about 6 to about 30 atoms in length.        In some embodiments, the X is selected from: DPRSFL (SEQ ID NO:        1), or PPRSFL (SEQ ID NO: 2). In some embodiments, M is a        macromolecular carrier selected from: a dendrimer, dextran, a        PEG polymer, albumin, or lipid-coated perfluorocarbon droplet.        In some embodiments, M is a PEG polymer. In some embodiments,        the imaging agent is selected from: a fluorescent moiety, a        luminescent moiety, a phosphorescent moiety, a        fluorescence-quenching moiety, a radioactive moiety, a        radiopaque moiety, a paramagnetic moiety, a contrast agent,        ultrasound scatterer, or a combination thereof. In some        embodiments, the imaging agent is selected from an        indocarbocyanine dye. In some embodiments, the imaging agent is        selected from Cy5, Cy5.5, Cy7, Alexa 647, IRDYE 800CW, or a        combination thereof. In some embodiments, the imaging agent is        an MRI contrast agent. In some embodiments, the imaging agent is        Gd complex of        [4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.

BRIEF DESCRIPTION OF THE DRAWINGS

The novel features of the invention are set forth with particularity inthe appended claims. A better understanding of the features andadvantages of the present invention will be obtained by reference to thefollowing detailed description that sets forth illustrative embodiments,in which the principles of the invention are utilized, and theaccompanying drawings of which:

FIG. 1. DPRSFL (SEQ ID NO: 1)-based transport molecule is cleaved bypurified thrombin and by clot-generated thrombin. A: Schematic of aselective transport molecule showing a positively charged polyarginineseparated from a negatively charged polyglutamine by a proteasecleavable linker. Once the linker is cleaved, the polyarginine is freeto associate with negatively charged membranes on cells, clots orplaques. B: Representative polyacrylamide gel showing cleavage of theDPRSFL (SEQ ID NO: 1)-based transport molecule by 50 nM thrombin (darkgrey) or 50% serum (light grey) after 20 minutes incubation. Directthrombin inhibitors were able to completely inhibit cleavage ofselective transport molecule. Experiments were done in triplicate. C:Representative polyacrylamide gel showing cleavage of DPRSFL (SEQ ID NO:1)-based transport molecule by several enzymes known to cleave the PAR-1receptor including trypsin, chymotrypsin, thrombin, plasmin and factorXa after one hour incubation in 50 nmol enzyme.

Experiments were done in triplicate.

FIG. 2. DPRSFL(SEQ ID NO: 1)-based transport molecule and PLGLAG (SEQ IDNO: 4)-selective transport molecule uptake are enzyme dependent andcorrelate with increased plaque burden in atherosclerotic mice. A-D:Representative fluorescence images of gross aortas removed from animalssix hours after injection with 10 nmol DPRSFL (SEQ ID NO: 1)-basedtransport molecule (A), 10 nmol DPRSFL (SEQ ID NO: 1)-based transportmolecule 45 minutes after injection with 250 μg hirudin (B), 10 nmolPLGLAG (SEQ ID NO: 4)-selective transport molecule and 10 nmol mPEGcontrol selective transport molecule (D), showing enzyme specificuptake. E-H: Fluorescence (E and G) and Gamori trichrome (F and H)micrographs of paraformaldehyde fixed plaques from animals injected withDPRSFL (SEQ ID NO: 1)-(E and F) and PLGLAG (SEQ ID NO: 4)-selectivetransport molecule (G and H). I: Scatter plot comparing average uptakeof DPRSFL (SEQ ID NO: 1)-based transport molecule in ApoE (solidsymbols) and LDLR (hollow symbols) deficient mice with the total plaqueburden, or percentage of each aorta covered in plaque. Uptake of theDPRSFL (SEQ ID NO: 1)-based transport molecule was largely inhibited inanimals pre-injected with hirudin, especially in aortas with increasedplaque burden.

FIG. 3. DPRSFL (SEQ ID NO: 1)-based transport molecule uptake correlateswith AHA class and rupture potential in ApoE and LDLR deficient mice.A-D: Light micrographs of H/E (A and C) and trichrome (B and D) stainedsections showing two representative lesions. E-F: Higher magnificationimages of the two sections in A and C. E: Category 5 plaque showinglipid pooling, an intact shoulder (blue arrow) and a thick fibrous cap(red arrows). F: Category 6 plaque displaying increased cellularity(yellow arrows) and an irregular contour indicative of an old thrombus.G-H: bar graphs comparing gross uptake of selective transport moleculewith the histopathological AHA class from a representative section.Uptake of the thrombin cleavable DPRSFL (SEQ ID NO: 1)-based transportmolecule correlates well with AHA class (G), whereas there is nosignificant difference between uptake of the MMP cleavable PLGLAG (SEQID NO: 4)-selective transport molecule in AHA Class 5 vs. AHA Class 6lesions (H).

FIG. 4. Dual selective transport molecule labeling of thrombin and MMPactivity in atherosclerosis. A and B: Gross fluorescence images fromanimals taken six hours after co-injection with rhodamine labeled DPRSFL(SEQ ID NO: 1)-based transport molecule (A) and Cy5 labeled PLGLAG (SEQID NO: 4)-selective transport molecule (B). Although both selectivetransport molecules highlight atheromas, uptake of DPRSFL (SEQ ID NO:1)-based transport molecule highlights specific regions of plaque (redarrow). C-E: Fluorescence histology images showing a small plaque on anotherwise clean vessel wall. The small plaque is highlighted with DPRSFL(SEQ ID NO: 1)-based transport molecule (C), but not with MMP-selectivetransport molecule (D), suggesting that thrombin may be a better targetfor visualization of early plaques. H/E staining is shown in (E). F-I:Cross sectional images of aorta showing DPRSFL (SEQ ID NO: 1)-basedtransport molecule uptake (F), PLGLAG (SEQ ID NO: 4)-selective transportmolecule uptake (G) and an overlay (H) with DPRSFL (SEQ ID NO: 1)-basedtransport molecule uptake shown in red and PLGLAG (SEQ ID NO:4)-selective transport molecule uptake shown in green in a slice of anadvanced plaque showing calcification (blue arrow) and fibrosis (redarrow) on an H/E (I). J-K: A magnified overlay of a difference slice ofaorta showing cells taking up DPRSFL (SEQ ID NO: 1)-based transportmolecule (green), PLGLAG (SEQ ID NO: 4)-selective transport molecule(red) or both (yellow). Again, the DPRSFL (SEQ ID NO: 1)-based transportmolecule also highlights areas of fibrosis (red arrows), identified byH/E staining (K).

FIG. 5. DPRSFL (SEQ ID NO: 1)-based transport molecule highlightsplaques in the carotid arteries of ApoE^(−/−) and LDLR^(−/−) mice. A andB: Brightfield (A) and Cy5 fluorescence micrographs of carotid plaquesin mice taken in living mice during a carotid endarterectomy proceduredone six hours after injection with I Onmol DPRSFL (SEQ ID NO: 1)-basedtransport molecule. Plaque barely visible in the intraoperative image(A) is highlighted in the fluorescence image (B). C-G: A fluorescencemicrograph taken postmortem of a similar animal following a moreextensive surgical exposure of the carotid bifurcation is shown in (C).From this specimen, vessel with (D-E) and without (F-G) highlightedplaques was taken for Cy5 fluorescence histology (D and F) and H/Estaining (E and G) that confirmed the presence (E) and absence (G) ofplaque. H-J: A similar fluorescence micrograph of a mouse co-injectedwith rhodamine labeled DPRSFL (SEQ ID NO: 1)-based transport molecule(H) and Cy5 labeled PLGLAG (SEQ ID NO: 4)-selective transport molecule(I) showing improved delineation of the plaque area with the DPRSFL (SEQID NO: 1)-based transport molecule. An H/E near the carotid bifurcationshowed near occlusion of the carotid artery (J).

FIG. 6. DPRSFL (SEQ ID NO: 1)-based transport molecule is taken up inhuman carotid endarterectomy specimen. A-C: Fluorescence micrographsshowing sections of atheromas incubated in DPRSFL (SEQ ID NO: 1)-basedtransport molecule (A), PLGLAG (SEQ ID NO: 4)-selective transportmolecule (B) or mPEG-selective transport molecule (C). The DPRSFL (SEQID NO: 1)-based transport molecule sections show the most uptake. D: Allof the atheromas tested were AHA Class 5 by H/E stain (D). E: Gelelectrophoresis confirming cleavage of the DPRSFL (SEQ ID NO: 1)-basedtransport molecule and PLGLAG (SEQ ID NO: 4)-selective transportmolecule but not the mPEG control selective transport molecule incarotid sections. F: Bar graph showing combined average intensities ofseven representative sections from each of the treatment groups (A-D).Statistical significance was assessed by unpaired t-test as indicated.

FIG. 7. DPRSFL (SEQ ID NO: 1) peptide is selectively cleaved by thrombinas well as by purified blood. (A) shows approximately 2.5 μM cleavablepeptide (Suc-e8-XDPRSFL-r9-c(cy5)) incubated for 20 minutes at roomtemperature under the indicated conditions. (B) shows the percentcleavage for the gel shown in (A). Similar results were seen for aSuc-e8-ODPRSFL-r9-c(cy5) analogue.

FIG. 8. Thrombin cleavable peptides are selectively taken up by specificcells in HT-1080 xenografts. (A) shows exposed tumors six hours postinjection with 10 nmol Suc-e8-XDPRSFL-r9-c(cy5). The all-d-amino acidanalogue is not cleavable by thrombin and therefore is not expected tobe taken up by tumors. (B) shows a cryosection of an HT-1080 tumor sixhours after injection with a Suc-e8-ODPRSFL-r9-c(cy5) peptide. Theidentity of the bright spots is unclear. Scale bar=1 mm.

FIG. 9. Thrombin cleavable peptides are selectively taken up by specificcells in B16-F10 melanoma syngeneic xenografts. (A) shows arepresentative section of tumor invading muscle from an animal injectedwith thrombin cleavable peptide. (B) shows standard hematoxylin/eosinstaining of the same slice. (C) and (D) show a similar region from ananimal injected with an uncleavable control peptide. Scale bars are 200μm.

FIG. 10. Uptake of thrombin cleavable peptide into lung metastases fromPyMT animals. Animals were injected with either the cleavable (A) oruncleavable (B) peptides and sacrificed six hours later.Hematoxylin/eosin stained adjacent slices (left) were used to findmetastases on frozen sections. Scale bar=200 μm.

FIG. 11. Very bright spots in lungs correspond to lymphocytes andmacrophages possibly surrounding tumor cells. Scale bars=100 μm.

FIG. 12. Uptake of Thrombin Cleavable Peptide into blood clots in vivo.This 180 mm long clot of unknown etiology was found in a lung from aPyMT mouse. In addition to lungs, brightly staining clots were foundinside vessels in tumors. Scale bar=200 μm.

FIG. 13. Thrombin peptide stains fresh clots, but not aged clots, in anin vitro clotting assay. (A) shows gross images of fresh clots incubatedfor ten minutes with either 3 or six μM of eitherSuc-e8-ODPRSFL-r9-c(cy5) or an all d-amino acid control probe with amPEG linker, then washed three times for ten minutes. (B) quantitatesthe results shown in (A) for three mice, two clots each.

FIG. 14. Uptake of thrombin probe is selective, but varies depending onthe size of the stroke.

FIG. 15. Dual color imaging verified the presence of a stroke in animalsinjected with the PEG-5 control peptide. (A) shows an animal thatreceived thrombin cleavable peptide labeled with cy5. (B) shows adifferent animal that was co-injected with cy5-labeled PEG-5 controlpeptide and rhodamine labeled thrombin cleavable peptide.

FIG. 16. Thrombin (Suc-e8-OPLGLAG-r9-c(cy5)) and plasmin(Suc-e8-XRLQLKL-r9-k(FITC)) cleavable selective transport molecules aretaken up selectively in an animal model for cerebral ischemia. An animalwas co-injected with thrombin (A) and (D) and plasmin (B) and (E)cleavable peptide immediately following reperfusion of the carotidartery. The two probes varied in distribution and did not colocalize(C). (F) shows a gross cy5 image of the same brain, cut sagittally andreflected outward.

FIG. 17. Thrombin cleavable selective transport molecules label small,500 μm clots caused by photooxidative damage. (A) shows vasculaturelabeled by FITC dextran. The clot caused by photoactivation of RoseBengal is shown by an arrow. (B) shows a cy5 image of the same field.(C) shows an overlay of (A) and (B). Scale bar=500 μm.

FIG. 18. Uptake of thrombin-cleavable, MMP-cleavable and PEG-5uncleavable selective transport molecules in atherosclerotic plaques inone year old female ApoE−/− mice.

FIG. 19. Uptake of thrombin-cleavable, MMP-cleavable and PEG-5uncleavable selective transport molecules in atherosclerotic plaques intwo year old male mice ApoE−/− mice. There is highlighted necrosispresent in the arch of the animal injected with the uncleavable controlprobe.

FIG. 20. Inhibition of thrombin-cleavable selective transport moleculeuptake in atherosclerotic plaques by hirudin and MMP-cleavable selectivetransport molecule uptake by an MMP inhibitor cocktail.

FIG. 21. Thrombin cleavable peptides can highlight regions ofatherosclerosis on MRI. This Figure shows a TI weighted MSME image ofthe internal carotid artery pre and post contrast. The arrow marks aregion of atherosclerotic plaques.

FIG. 22. A: DPRSFL (SEQ ID NO: 1)-selective transport molecules andPPRSFL(SEQ ID NO: 2)-selective transport molecule cleavage after onehour incubation in a panel of commercially available enzymes showingnear complete cleavage by trypsin, chymotrypsin, thrombin, plasmin andFactor Xa. B: Quantitative comparison of thrombin, plasmin and factor Xacleavage of DPRSFL (SEQ ID NO: 1)- and PPRSFL (SEQ ID NO: 2)-selectivetransport molecules after 15 minutes of incubation.

FIG. 23. DPRSFL (SEQ ID NO: 1)- and PPRSFL (SEQ ID NO: 2)-selectivetransport molecules are subject to proteolytic cleavage by endogenousproteases in serum. A: DPRSFL (SEQ ID NO: 1)-selective transportmolecules and PPRSFL (SEQ ID NO: 2)-selective transport molecules aftera 20 minute incubation in serum, serum plus direct thrombin inhibitor orplasma as indicated. Data are expressed as % increase over control, orselective transport molecules alone. B: shows a time course showingslower cleavage of PPRSFL (SEQ ID NO: 2)-selective transport moleculesthan DPRSFL (SEQ ID NO: 1)-selective transport molecules in serum. Eachcurve was fit to an exponential, which revealed a 51% drop in rateconstant for PPRSFL (SEQ ID NO: 2)-selective transport moleculesrelative to DPRSFL (SEQ ID NO: 1)-selective transport molecules.

FIG. 24. Both DPRSFL (SEQ ID NO: 1) and PPRSFL (SEQ ID NO: 2) arecleaved by coagulation pathway proteases. Top panel: (1) Sensitivity ofequimolar concentrations of DPRSFL (SEQ ID NO: 1) and PPRSFL (SEQ ID NO:2) to enzymatic cleavage across a range of thrombin concentrations. BothDPRSFL (SEQ ID NO: 1) and PPRSFL (SEQ ID NO: 2) are cleaved by thrombinin vitro as shown by the lower molecular weight band (“cleaved”)observed after incubation with 50 nM thrombin. Lower concentrations ofthrombin (from 0.1 nM to 5 nM) results in inefficient cleavage after 40minutes of incubation. Middle Panel (2): Comparison of the relativespecificity of DPRSFL (SEQ ID NO: 1) and PPRSFL (SEQ ID NO: 2) cleavagewith a panel of coagulation pathway enzymes. Cleavage was assessed after15 minutes of enzyme-ACPP incubation. Cleavage of PPRSFL (SEQ ID NO: 2)is relatively selective for thrombin while DPRSFL (SEQ ID NO: 1) alsoshows cleavage products when incubated with plasmin and factor XA.

FIG. 25. Coinjection of DPRSFL (SEQ ID NO: 1) (Cy5 labeled) and ofPPRSFL (SEQ ID NO: 2) (Rhodamine labeled) into mice bearing BI6F10melanoma tumors demonstrates similar patterns of fluorescence. Toppanel: Control experiment to test the colocalization of fluorescencefrom injections of DPRSFL labeled with Cy5 and of DPRSFL (SEQ ID NO: 1)labeled with Rhodamine. The left hand and middle panels showsfluorescence in whole tumor imaged with a Zeiss Lumar microscope afterthe skin was removed. The first image is of inverted fluorescence tobetter illustrate the heterogeneous uptake pattern. Similarheterogeneity is observed in the same animal with uptake of DPRSFL (SEQID NO: 1) labeled with rhodamine (extreme right hand panel). MiddlePanel: Skin-off images of tumor fluorescence of PPRSFL-(Cy5) (SEQ ID NO:5) and of DPRSFL-(Rhodamine) (SEQ ID NO: 6). The uptake pattern isgrossly similar. Bottom Panel: Skin-on images also show similar tumorfluorescence with DPRSFL (SEQ ID NO: 1) and PPRSFL (SEQ ID NO: 2) aftercoinjection of PPRSFL-Cy5 (SEQ ID NO: 5) and of DPRSFL-Rhodamine (SEQ IDNO: 6).

FIG. 26. FIG. 3. PPRSFL-Cy5 (SEQ ID NO: 5) labels the adult mouse brainafter experimental stroke. Injured vasculature on the base of an adultmouse brain after transient unilateral occlusion of the internal carotidartery with a nylon filament is labeled with PPRSFL-Cy5 (SEQ ID NO: 5).(Similar focal accumulations of DPRSFL (SEQ ID NO: 1) fluorescence havebeen observed with this filament-artery occlusion model of stroke in theadult rat.) Left panel illustrates the base of a mouse brain after a 2hour stroke from transient blockage of the internal carotid artery witha withdrawable nylon filament. PPRSFL-Cy5 (SEQ ID NO: 5) was injected atthe start of the 6 hour reperfusion period. Cy5 fluorescenceconcentrates with the injured artery and also at a second site situatedin the brain parenchyma near the base of the ostium of the middlecerebral artery. Middle panel shows a view of the base of the brainwhere the fluorescence is inverted (and appears as a dark signal). Rightpanel is illustrates vascular injury from this filament occlusion toverify an ischemic stroke. In this case the filament occlusion appearsto have caused a hemorrhage and blood efflux onto the surface of thebrain.

DETAILED DESCRIPTION OF THE INVENTION

Disclosed herein, in certain embodiments, is a selective transportmolecule.

In some embodiments, a selective transport molecule disclosed herein hasthe formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with asequence comprising 5 to 9 consecutive acidic amino acids, wherein theamino acids are selected from: aspartates and glutamates; B is a peptidewith a sequence comprising 5 to 20 consecutive basic amino acids; and Xis a linker that is cleavable by thrombin.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B-C)_(n)-M, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; and M is amacromolecular carrier.

Certain Definitions

The following terms have the meanings ascribed to them unless specifiedotherwise.

The terms cell penetrating peptide (CPP), membrane translocatingsequence (MTS) and protein transduction domain are used interchangeably.As used herein, the terms mean a peptide (polypeptide or protein)sequence that is able to translocate across the plasma membrane of acell. In some embodiments, a CPP facilitates the translocation of anextracellular molecule across the plasma membrane of a cell. In someembodiments, the CPP translocates across the plasma membrane by directpenetration of the plasma membrane, endocytosis-mediated entry, or theformation of a transitory structure.

As used herein, the term “aptamer” refers to a DNA or RNA molecule thathas been selected from random pools based on their ability to bind othermolecules with high affinity specificity based on non-Watson and Crickinteractions with the target molecule (see, e.g., Cox and Ellington,Bioorg. Med. Chem. 9:2525-2531 (2001); Lee et al., Nuc. Acids Res.32:D95-D100 (2004)). In some embodiments, the aptamer binds nucleicacids, proteins, small organic compounds, vitamins, inorganic compounds,cells, and even entire organisms.

The terms “polypeptide,” “peptide” and “protein” are usedinterchangeably herein to refer to a polymer of amino acid residues. Theterms apply to naturally occurring amino acid polymers as well as aminoacid polymers in which one or more amino acid residues is anon-naturally occurring amino acid (e.g., an amino acid analog). Theterms encompass amino acid chains of any length, including full lengthproteins (i.e., antigens), wherein the amino acid residues are linked bycovalent peptide bonds. As used herein, the terms “peptide” refers to apolymer of amino acid residues typically ranging in length from 2 toabout 50 residues. In certain embodiments the peptide ranges in lengthfrom about 2, 3, 4, 5, 7, 9, 10, or 11 residues to about 50, 45, 40, 45,30, 25, 20, or 15 residues. In certain embodiments the peptide ranges inlength from about 8, 9, 10, 11, or 12 residues to about 15, 20 or 25residues. Where an amino acid sequence is provided herein, L-, D-, orbeta amino acid versions of the sequence are also contemplated as wellas retro, inversion, and retro-inversion isoforms. Peptides also includeamino acid polymers in which one or more amino acid residues is anartificial chemical analogue of a corresponding naturally occurringamino acid, as well as to naturally occurring amino acid polymers. Inaddition, the term applies to amino acids joined by a peptide linkage orby other modified linkages (e.g., where the peptide bond is replaced byan a-ester, a 13-ester, a thioamide, phosphonamide, carbamate,hydroxylate, and the like (see, e.g., Spatola, (1983) Chem. Biochem.Amino Acids and Proteins 7: 267-357), where the amide is replaced with asaturated amine (see, e.g., Skiles et al., U.S. Pat. No. 4,496,542,which is incorporated herein by reference, and Kaltenbronn et al.,(1990) Pp. 969-970 in Proc. 11th American Peptide Symposium, ESCOMScience Publishers, The Netherlands, and the like)).

The term “amino acid” refers to naturally occurring and synthetic aminoacids, as well as amino acid analogs and amino acid mimetics thatfunction in a manner similar to the naturally occurring amino acids.Naturally occurring amino acids are those encoded by the genetic code,as well as those amino acids that are later modified, e.g.,hydroxyproline, γ-carboxyglutamate, and O-phosphoserine. Amino acidanalogs refers to compounds that have the same basic chemical structureas a naturally occurring amino acid, i.e., an a carbon that is bound toa hydrogen, a carboxyl group, an amino group, and an R group, e.g.,homoserine, norleucine, methionine sulfoxide. Such analogs have modifiedR groups (e.g., norleucine) or modified peptide backbones, but retainthe same basic chemical structure as a naturally occurring amino acid.Amino acid mimetics refers to chemical compounds that have a structurethat is different from the general chemical structure of an amino acid,but that functions in a manner similar to a naturally occurring aminoacid. Amino acids may be either D amino acids or L amino acids. Inpeptide sequences throughout the specification, lower case lettersindicate the D isomer of the amino acid (conversely, upper case lettersindicate the L isomer of the amino acid).

Amino acids may be referred to herein by either their commonly knownthree letter symbols or by the one-letter symbols recommended by theIUPAC-IUB Biochemical Nomenclature Commission. Nucleotides, likewise,may be referred to by their commonly accepted single-letter codes.

One of skill will recognize that individual substitutions, deletions oradditions to a peptide, polypeptide, or protein sequence which alters,adds or deletes a single amino acid or a small percentage of amino acidsin the encoded sequence is a “conservatively modified variant” where thealteration results in the substitution of an amino acid with achemically similar amino acid. Conservative substitution tablesproviding functionally similar amino acids are well known in the art.Such conservatively modified variants are in addition to and do notexclude polymorphic variants, interspecies homologs, and alleles of theinvention.

The following eight groups each contain amino acids that areconservative substitutions for one another: 1) Alanine (A), Glycine (G);2) Aspartic acid (D), Glutamic acid (E); 3) Asparagine (N), Glutamine(Q); 4) Arginine (R), Lysine (K); 5) Isoleucine (I), Leucine (L),Methionine (M), Valine (V); 6) Phenylalanine (F), Tyrosine (Y),Tryptophan (W); 7) Serine (S), Threonine (T); and 8) Cysteine (C),Methionine (M) (see, e.g., Creighton, Proteins (1984)).

As used herein, a “linker” is any molecule capable of binding (e.g.,covalently) portion A and portion B of a selective transport moleculedisclosed herein. Linkers include, but are not limited to, straight orbranched chain carbon linkers, heterocyclic carbon linkers, peptidelinkers, and polyether linkers. For example, poly(ethylene glycol)linkers are available from Quanta Biodesign, Powell, Ohio. These linkersoptionally have amide linkages, sulfhydryl linkages, or heterofunctionallinkages.

As used herein, the term “label” refers to any molecule that facilitatesthe visualization and/or detection of a selective transport moleculedisclosed herein. In some embodiments, the label is a fluorescentmoiety.

The term “carrier” means an inert molecule that increases (a) plasmahalf-life and (b) solubility. In some embodiments, a carrier increasesplasma half-life and solubility by reducing glomerular filtration. Insome embodiments, a carrier increases tumor uptake due to enhancedpermeability and retention (EPR) of tumor vasculature.

The term “thrombin” means an enzyme (EC 3.4.21.5) that cleavesfibrinogen molecules into fibrin monomers. Thrombin, acting through itsG-protein coupled receptor PAR-1, is a key player in a wide range ofvascular and extravascular disease processes throughout the body,including cancer, cardiovascular diseases, acute kidney injury, andstroke. In certain instances, thrombin activity increases over thecourse of atherosclerotic plaque development. In some embodiments,thrombin activity is a biomarker for atherosclerotic plaque development.

The terms “individual,” “patient,” or “subject” are usedinterchangeably. As used herein, they mean any mammal (i.e. species ofany orders, families, and genus within the taxonomic classificationanimalia: chordata: vertebrata: mammalia). In some embodiments, themammal is a human. None of the terms require or are limited to situationcharacterized by the supervision (e.g. constant or intermittent) of ahealth care worker (e.g. a doctor, a registered nurse, a nursepractitioner, a physician's assistant, an orderly, or a hospice worker).

As used herein, the term “medical professional” means any health careworker. By way of non-limiting example, the health care worker may be adoctor, a registered nurse, a nurse practitioner, a physician'sassistant, an orderly, or a hospice worker.

The terms “administer,” “administering”, “administration,” and the like,as used herein, refer to the methods that may be used to enable deliveryof agents or compositions to the desired site of biological action.These methods include, but are not limited to parenteral injection(e.g., intravenous, subcutaneous, intraperitoneal, intramuscular,intravascular, intrathecal, intravitreal, infusion, or local).Administration techniques that are optionally employed with the agentsand methods described herein, include e.g., as discussed in Goodman andGilman, The Pharmacological Basis of Therapeutics, current ed.;Pergamon; and Remington's, Pharmaceutical Sciences (current edition),Mack Publishing Co., Easton, Pa.

The term “pharmaceutically acceptable” as used herein, refers to amaterial that does not abrogate the biological activity or properties ofthe agents described herein, and is relatively nontoxic (i.e., thetoxicity of the material significantly outweighs the benefit of thematerial). In some instances, a pharmaceutically acceptable material maybe administered to an individual without causing significant undesirablebiological effects or significantly interacting in a deleterious mannerwith any of the components of the composition in which it is contained.

The term “surgery” as used herein, refers to any methods for that may beused to manipulate, change, or cause an effect by a physicalintervention. These methods include, but are not limited to opensurgery, endoscopic surgery, laparoscopic surgery, minimally invasivesurgery, and robotic surgery

The following symbols, where used, are used with the indicated meanings:Fl=fluorescein; aca=ahx=X=aminohexanoyl linker(—HN—((CH₂)₅—CO-)aminohexanoyl; C=L-cysteine; E=L-glutamate;R=L-arginine; D=L-aspartate; K=L-lysine; A=L-alanine; r=D-arginine;c=D-cysteine; e=D-glutamate; P=L-proline; L=L-leucine; G=glycine;V=valine; I=isoleucine; M=methionine; F=phenylalanine; Y=tyrosine;W=tryptophan; H=histidine; Q=glutamine; N=asparagine; S=serine;T=threonine, o is 5-amino-3-oxapentanoyl linker, and C(me) isS-methylcysteine.

Selective Transport Molecules

Regulation of transport into and out of a cell is important for itscontinued viability. For example, cell membranes contain ion channels,pumps, and exchangers capable of facilitating the transmembrane passageof many important substances. However, transmembrane transport isselective: in addition to facilitating the entry of desired substancesinto a cell, and facilitating the exit of others, a major role of a cellmembrane is to prevent uncontrolled entry of substances into the cellinterior. This barrier function of the cell membrane makes difficult thedelivery of markers, drugs, nucleic acids, and other exogenous materialinto cells.

Multiple membrane translocation signals (MTS) have been identified. Forexample, the Tat protein of the human immunodeficiency virus 1 (HIV-1)is able to enter cells from the extracellular environment. A domain fromAntennapedia homeobox protein is also able to enter cells.

Molecules comprising a MTS may also be used to carry other moleculesinto cells along with them. The most important MTS are rich in aminoacids such as arginine with positively charged side chains. Moleculestransported into cell by such cationic peptides may be termed “cargo”and may be reversibly or irreversibly linked to the cationic peptides.

The uptake facilitated by molecules comprising a MTS is currentlywithout specificity, enhancing uptake into most or all cells. It isdesirable to have the ability to target the delivery of cargo to a typeof cell, or to a tissue, or to a location or region within the body ofan animal. Accordingly, we have identified a need for a selectivetransport molecule with increased in vivo circulation.

In some embodiments, a selective transport molecule disclosed herein hasthe formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with asequence comprising 5 to 9 consecutive acidic amino acids, wherein theamino acids are selected from: aspartates and glutamates; B is a peptidewith a sequence comprising 5 to 20 consecutive basic amino acids; and Xis a linker that is cleavable by thrombin.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B-C)_(n)-M, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; M is amacromolecular carrier; and n is an integer between 1 and 20.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B)_(n)-D, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; D is a dendrimer;and n is an integer between 1 and 20. In some embodiments, D comprises acargo moiety.

In embodiments, a selective transport molecule disclosed herein is alinear molecule. In embodiments, a selective transport moleculedisclosed herein is a cyclic molecule, as schematically illustrated inFIG. 1B. In embodiments, a selective transport molecule disclosed hereincomprises a cyclic portion and a linear portion.

A selective transport molecule disclosed herein may be of any length. Insome embodiments, a selective transport molecule disclosed herein isabout 7 to about 40 amino acids in length, not including the length of alinker X and a cargo moiety C. In other embodiments, particularly wheremultiple non-acidic (in portion A) or non-basic (in portion B) aminoacids are included in one or both of portions A and B, portions A and Bof a selective transport molecule disclosed herein may together be about50, or about 60, or about 70 amino acids in length. A cyclic portion ofa selective transport molecule disclosed herein may include about 12 toabout 60 amino acids, not including the length of a linker X and a cargomoiety C. For example, a linear selective transport molecule disclosedherein may have a basic portion B having between about 5 to about 20basic amino acids (preferably between about 9 to about 16 basic aminoacids) and an acidic portion A having between about 2 to about 20 acidicamino acids (e.g., between about 5 to about 20, preferably between about5 to about 9 acidic amino acids). In some preferred embodiments, aselective transport molecule disclosed herein may have a basic portion Bhaving between about 9 to about 16 basic amino acids and between about 5to about 9 acidic amino acids. In some embodiments, A is 8 consecutiveglutamates (i.e., EEEEEEEE, E₉, eeeeeeee, or e9), B is nine consecutivearginines (i.e., RRRRRRRRR, R₉, rrrrrrrrr, or r9), and X is PLGLAG.

In some embodiments, the selective transport molecule is selected from:Suc-e₉-XDPRSFL-r₉-c(Cy5)-CONH₂; Suc-e₉-ODPRSFL-r₉-c(Cy5)-CONH₂; andSuc-e₉-Xdprsfl-r₉-c(Cy5)-CONH₉.

Peptide Synthesis

A selective transport molecule disclosed herein is synthesized by anysuitable method, such as, for example, solid phase synthesis includingsolid phase peptide synthesis. An example of peptide synthesis usingFmoc is given as Example 1 below. For example, conventional solid phasemethods for synthesizing peptides may start with N-alpha-protected aminoacid anhydrides that are prepared in crystallized form or preparedfreshly in solution, and are used for successive amino acid addition atthe N-terminus. At each residue addition, the growing peptide (on asolid support) is acid treated to remove the N-alpha-protective group,washed several times to remove residual acid and to promoteaccessibility of the peptide terminus to the reaction medium. Thepeptide is then reacted with an activated N-protected amino acidsymmetrical anhydride, and the solid support is washed. At eachresidue-addition step, the amino acid addition reaction may be repeatedfor a total of two or three separate addition reactions, to increase thepercent of growing peptide molecules which are reacted. Typically, 1 to2 reaction cycles are used for the first twelve residue additions, and 2to 3 reaction cycles for the remaining residues.

After completing the growing peptide chains, the protected peptide resinis treated with a strong acid such as liquid hydrofluoric acid ortrifluoroacetic acid to deblock and release the peptides from thesupport. For preparing an amidated peptide, the resin support used inthe synthesis is selected to supply a C-terminal amide, after peptidecleavage from the resin. After removal of the strong acid, the peptidemay be extracted into 1M acetic acid solution and lyophilized. Thepeptide may be isolated by an initial separation by gel filtration, toremove peptide dimers and higher molecular weight polymers, and also toremove undesired salts The partially purified peptide may be furtherpurified by preparative HPLC chromatography, and the purity and identityof the peptide confirmed by amino acid composition analysis, massspectrometry and by analytical HPLC (e.g., in two different solventsystems).

Polynucleotide Synthesis

Disclosed herein, in certain embodiments is a polynucleotide encoding aselective transport molecule described herein. The term “polynucleotide”refers to a polymeric form of nucleotides of at least 10 bases inlength. Nucleotides include ribonucleotides, deoxynucleotides, ormodified forms of either type of nucleotide. The term includes singleand double stranded forms of DNA. The term therefore includes, forexample, a recombinant DNA which is incorporated into a vector, e.g., anexpression vector; into an autonomously replicating plasmid or virus; orinto the genomic DNA of a prokaryote or eukaryote, or which exists as aseparate molecule (e.g., a cDNA) independent of other sequences.

These polynucleotides include DNA, cDNA, and RNA sequences which encodea selective transport molecule described herein, or portions thereof. Insome embodiments, polynucleotides include promoter and other sequences,and may be incorporated into a vector for transfection (which may bestable or transient) in a host cell.

The construction of expression vectors and the expression of genes intransfected cells involves the use of molecular cloning techniques thatare well known in the art. See, for example, Sambrook et al., MolecularCloning—A Laboratory Manual, Cold Spring Harbor Laboratory, Cold SpringHarbor, N.Y., (1989) and Current Protocols in Molecular Biology, F. M.Ausubel et al., eds., (Current Protocols, a joint venture between GreenePublishing Associates, Inc. and John Wiley & Sons, Inc., most recentSupplement). Nucleic acids used to transfect cells with sequences codingfor expression of the polypeptide of interest generally will be in theform of an expression vector including expression control sequencesoperatively linked to a nucleotide sequence coding for expression of thepolypeptide. As used herein, “operatively linked” refers to ajuxtaposition wherein the components so described are in a relationshippermitting them to function in their intended manner. A control sequenceoperatively linked to a coding sequence is ligated such that expressionof the coding sequence is achieved under conditions compatible with thecontrol sequences. “Control sequence” refers to polynucleotide sequenceswhich are necessary to effect the expression of coding and non-codingsequences to which they are ligated. Control sequences generally includepromoter, ribosomal binding site, and transcription terminationsequence. The term “control sequences” is intended to include, at aminimum, components that are able to influence expression, and alsoinclude additional components whose presence is advantageous, forexample, leader sequences and fusion partner sequences. As used herein,the term “nucleotide sequence coding for expression or a polypeptiderefers to a sequence that, upon transcription and translation of mRNA,produces the polypeptide. This includes sequences containing, e.g.,introns. As used herein, the term “expression control sequences” refersto nucleic acid sequences that regulate the expression of a nucleic acidsequence to which it is operatively linked. Expression control sequencesare operatively linked to a nucleic acid sequence when the expressioncontrol sequences control and regulate the transcription and, asappropriate, translation of the nucleic acid sequence. Thus, expressioncontrol sequences include appropriate promoters, enhancers,transcription terminators, a start codon (i.e., ATG) in front of aprotein-encoding gene, splicing signals for introns, maintenance of thecorrect reading frame of that gene to permit proper translation of themRNA, and stop codons.

Any suitable method is used to construct expression vectors containingthe fluorescent indicator coding sequence and appropriatetranscriptional/translational control signals. These methods include invitro recombinant DNA techniques, synthetic techniques and in vivorecombination/genetic recombination. (See, for example, the techniquesdescribed in Maniatis, et al., Molecular Cloning A Laboratory Manual,Cold Spring Harbor Laboratory, N.Y., 1989). Transformation of a hostcell with recombinant DNA may be carried out by conventional techniquesas are well known to those skilled in the art.

Where the host is prokaryotic, such as E. coli, competent cells (i.e.,cell which are capable of DNA uptake) are prepared from cells harvestedafter exponential growth phase and subsequently treated with CaCl₂,MgCl₂ or RbCl. In certain instances, transformation is performed afterforming a protoplast of the host cell or by electroporation.

When the host is a eukaryote, such methods of transfection of DNA ascalcium phosphate co-precipitates, conventional mechanical proceduressuch as microinjection, electroporation, insertion of a plasmid encasedin Liposomes, or virus vectors may be used. In certain instances,eukaryotic cells are co-transfected with DNA sequences encoding amolecule disclosed herein, and a second foreign DNA molecule encoding aselectable phenotype, such as the herpes simplex thymidine kinase gene.Another method is to use a eukaryotic viral vector, such as simian virus40 (SV40) or bovine papilloma virus, to transiently infect or transformeukaryotic cells and express the protein. (Eukaryotic Viral Vectors,Cold Spring Harbor Laboratory, Gluzman ed., 1982). Techniques for theisolation and purification of polypeptides of the invention expressed inprokaryotes or eukaryotes may be by any conventional means such as, forexample, preparative chromatographic separations and immunologicalseparations such as those involving the use of monoclonal or polyclonalantibodies or antigen.

Portion A

Disclosed herein, in certain embodiments, is a selective transportmolecule.

In some embodiments, a selective transport molecule disclosed herein hasthe formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with asequence comprising 5 to 9 consecutive acidic amino acids, wherein theamino acids are selected from: aspartates and glutamates; B is a peptidewith a sequence comprising 5 to 20 consecutive basic amino acids; and Xis a linker that is cleavable by thrombin.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B-C)_(n)-M, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; M is amacromolecular carrier; and n is an integer between 1 and 20.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B)_(n)-D, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; D is a dendrimer;and n is an integer between 1 and 20. In some embodiments, D comprises acargo moiety. See Example 1 for methods of attaching a label to aselective transport molecule.

In some embodiments of molecules having features of the invention,peptide portion A includes between about 2 to about 20, or between about5 to about 20 acidic amino acids, and may be series of acidic aminoacids (e.g., glutamates and aspartates or other acidic amino acids). Insome embodiments, A has a sequence comprising 5 to 9 consecutiveglutamates.

In some embodiments, portion A comprises 8 consecutive glutamates (i.e.,EEEEEEEE or eeeeeeee).

An acidic portion A may include amino acids that are not acidic. Acidicportion A may comprise other moieties, such as negatively chargedmoieties. In embodiments of a selective transport molecule disclosedherein, an acidic portion A may be a negatively charged portion,preferably having about 2 to about 20 negative charges at physiologicalpH that does not include an amino acid. In preferred embodiments, theamount of negative charge in portion A is approximately the same as theamount of positive charge in portion B.

Portion A is either L-amino acids or D-amino acids. In embodiments ofthe invention, D-amino acids are preferred in order to minimizeimmunogenicity and nonspecific cleavage by background peptidases orproteases. Cellular uptake of oligo-D-arginine sequences is known to beas good as or better than that of oligo-L-arginines.

It will be understood that portion A may include non-standard aminoacids, such as, for example, hydroxylysine, desmosine, isodesmosine, orother non-standard amino acids. Portion A may include modified aminoacids, including post-translationally modified amino acids such as, forexample, methylated amino acids (e.g., methyl histidine, methylatedforms of lysine, etc.), acetylated amino acids, amidated amino acids,formylated amino acids, hydroxylated amino acids, phosphorylated aminoacids, or other modified amino acids. Portion A may also include peptidemimetic moieties, including portions linked by non-peptide bonds andamino acids linked by or to non-amino acid portions.

The generic structures A-X-B and -A-X-B-C is effective where A is at theamino terminus or where A is at the carboxy terminus, i.e., eitherorientation of the peptide bonds is permissible.

Portion B (Membrane-Translocatinz Sequence)

Disclosed herein, in certain embodiments, is a selective transportmolecule.

In some embodiments, a selective transport molecule disclosed herein hasthe formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with asequence comprising 5 to 9 consecutive acidic amino acids, wherein theamino acids are selected from: aspartates and glutamates; B is a peptidewith a sequence comprising 5 to 20 consecutive basic amino acids; and Xis a linker that is cleavable by thrombin.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B-C)_(n)-M, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; M is amacromolecular carrier; and n is an integer between 1 and 20.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B)_(n)-D, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; D is a dendrimer;and n is an integer between 1 and 20. In some embodiments, D comprises acargo moiety. See Example 1 for methods of attaching a label to aselective transport molecule.

In some embodiments of molecules having features of the invention,peptide portion B includes between about 5 to about 20, or between about9 to about 16 basic amino acids, and may be a series of basic aminoacids (e.g., arginines, histidines, lysines, or other basic aminoacids).

In some embodiments, portion B comprises 9 consecutive arginines (i.e.,RRRRRRRRR or rrrrrrrrr).

A basic portion B may include amino acids that are not basic. Basicportion B may comprise other moieties, such as positively chargedmoieties. In embodiments, a basic portion B may be a positively chargedportion, preferably having between about 5 and about 20 positive chargesat physiological pH, that does not include an amino acid. In preferredembodiments, the amount of negative charge in portion A is approximatelythe same as the amount of positive charge in portion B.

Portion B is either L-amino acids or D-amino acids. In embodiments ofthe invention, D-amino acids are preferred in order to minimizeimmunogenicity and nonspecific cleavage by background peptidases orproteases. Cellular uptake of oligo-D-arginine sequences is known to beas good as or better than that of oligo-L-arginines.

It will be understood that portion B may include non-standard aminoacids, such as, for example, hydroxylysine, desmosine, isodesmosine, orother non-standard amino acids. Portion B may include modified aminoacids, including post-translationally modified amino acids such as, forexample, methylated amino acids (e.g., methyl histidine, methylatedforms of lysine, etc.), acetylated amino acids, amidated amino acids,formylated amino acids, hydroxylated amino acids, phosphorylated aminoacids, or other modified amino acids. Portion B may also include peptidemimetic moieties, including portions linked by non-peptide bonds andamino acids linked by or to non-amino acid portions.

In embodiments where X is a peptide cleavable by a protease, it may bepreferable to join the C-terminus of X to the N-terminus of B, so thatthe new amino terminus created by cleavage of X contributes anadditional positive charge that adds to the positive charges alreadypresent in B.

Portion X (Linkers)

Disclosed herein, in certain embodiments, is a selective transportmolecule.

In some embodiments, a selective transport molecule disclosed herein hasthe formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with asequence comprising 5 to 9 consecutive acidic amino acids, wherein theamino acids are selected from: aspartates and glutamates; B is a peptidewith a sequence comprising 5 to 20 consecutive basic amino acids; and Xis a linker that is cleavable by thrombin.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B-C)_(n)-M, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; M is amacromolecular carrier; and n is an integer between 1 and 20.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B)_(n)-D, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; D is a dendrimer;and n is an integer between 1 and 20. In some embodiments, D comprises acargo moiety. See Example 1 for methods of attaching a label to aselective transport molecule.

Cleavage Conditions

In some embodiments, X is a cleavable linker. In some embodiments, alinker X is designed for cleavage in the presence of particularconditions or in a particular environment. In preferred embodiments, alinker X is cleavable under physiological conditions. Cleavage of such alinker X may, for example, be enhanced or may be affected by particularpathological signals or a particular environment related to cells inwhich cargo delivery is desired. The design of a linker X for cleavageby specific conditions, such as by a specific enzyme (e.g., thrombin),allows the targeting of cellular uptake to a specific location wheresuch conditions obtain. Thus, one important way that selective transportmolecules provide specific targeting of cellular uptake to desiredcells, tissues, or regions is by the design of the linker portion X tobe cleaved by conditions near such targeted cells, tissues, or regions.After cleavage of a linker X, the portions B-C of the molecule are thena simple conjugate of B and C, in some instances retaining a relativelysmall, inert stub remaining from a residual portion of linker X.

In some embodiments, X is cleaved by thrombin. In some embodiments, X issubstantially specific for thrombin.

Linkers

In some embodiments, a linker consisting of one or more amino acids isused to join peptide sequence A (i.e., the sequence designed to preventuptake into cells) and peptide sequence B (i.e., the MTS). Generally thepeptide linker will have no specific biological activity other than tojoin the molecules or to preserve some minimum distance or other spatialrelationship between them. However, the constituent amino acids of thelinker may be selected to influence some property of the molecule suchas the folding, net charge, or hydrophobicity.

In some embodiments, X is a cleavable linker.

In some embodiments, the linker is flexible. In some embodiments, thelinker is rigid.

In some embodiments, the linker comprises a linear structure. In someembodiments, the linker comprises a non-linear structure. In someembodiments, the linker comprises a branched structure. In someembodiments, the linker comprises a cyclic structure.

In some embodiments, X is about 5 to about 30 atoms in length. In someembodiments, X is about 6 atoms in length. In some embodiments, X isabout 8 atoms in length. In some embodiments, X is about 10 atoms inlength. In some embodiments, X is about 12 atoms in length. In someembodiments, X is about 14 atoms in length. In some embodiments, X isabout 16 atoms in length. In some embodiments, X is about 18 atoms inlength. In some embodiments, X is about 20 atoms in length. In someembodiments, X is about 25 atoms in length. In some embodiments, X isabout 30 atoms in length.

In some embodiments, X is cleaved by thrombin. In some embodiments, thelinker is substantially specific for thrombin.

In some embodiments, the linker has a formula selected from: DPRSFL (SEQID NO: 1), or PPRSFL (SEQ ID NO: 2).

In some embodiments, the linker binds peptide portion A (i.e., thepeptide sequence which prevents cellular uptake) to peptide portion B(i.e., the MTS sequence) by a covalent linkage. In some embodiments, thecovalent linkage comprises an ether bond, thioether bond, amine bond,amide bond, carbon-carbon bond, carbon-nitrogen bond, carbon-oxygenbond, or carbon-sulfur bond.

In some embodiments, X comprises a peptide linkage. The peptide linkagecomprises L-amino acids and/or D-amino acids. In embodiments of theinvention, D-amino acids are preferred in order to minimizeimmunogenicity and nonspecific cleavage by background peptidases orproteases. Cellular uptake of oligo-D-arginine sequences is known to beas good as or better than that of oligo-L-arginines.

It will be understood that a linker disclosed herein may includenon-standard amino acids, such as, for example, hydroxylysine,desmosine, isodesmosine, or other non-standard amino acids. A linkerdisclosed herein may include modified amino acids, includingpost-translationally modified amino acids such as, for example,methylated amino acids (e.g., methyl histidine, methylated forms oflysine, etc.), acetylated amino acids, amidated amino acids, formylatedamino acids, hydroxylated amino acids, phosphorylated amino acids, orother modified amino acids. A linker disclosed herein may also includepeptide mimetic moieties, including portions linked by non-peptide bondsand amino acids linked by or to non-amino acid portions.

In some embodiments, a selective transport molecule disclosed hereincomprises a single of linker. Use of a single mechanism to mediateuptake of both imaging and therapeutic cargoes is particularly valuable,because imaging with noninjurious tracer quantities can be used to testwhether a subsequent therapeutic dose is likely to concentrate correctlyin the target tissue.

In some embodiments, a selective transport molecule disclosed hereincomprises a plurality of linkers. Where a selective transport moleculedisclosed herein includes multiple linkages X, separation of portion Afrom the other portions of the molecule requires cleavage of alllinkages X. Cleavage of multiple linkers X may be simultaneous orsequential. Multiple linkages X may include linkages X having differentspecificities, so that separation of portion A from the other portionsof the molecule requires that more than one condition or environment(“extracellular signals”) be encountered by the molecule. Cleavage ofmultiple linkers X thus serves as a detector of combinations of suchextracellular signals. For example, a selective transport molecule mayinclude two linker portions Xa and Xb connecting basic portion B withacidic portion A. Both linkers Xa and Xb must be cleaved before acidicportion A is separated from basic portion B allowing entry of portion Band cargo moiety C (if any) to enter a cell. It will be understood thata linker region may link to either a basic portion B or a cargo moiety Cindependently of another linker that may be present, and that, wheredesired, more than two linker regions X may be included.

Combinations of two or more linkers X may be used to further modulatethe targeting and delivery of molecules to desired cells, tissue orregions. Combinations of extracellular signals are used to widen ornarrow the specificity of the cleavage of linkers X if desired. Wheremultiple linkers X are linked in parallel, the specificity of cleavageis narrowed, since each linker X must be cleaved before portion A mayseparate from the remainder of the molecule. Where multiple linkers Xare linked in series, the specificity of cleavage is broadened, sincecleavage on any one linker X allows separation of portion A from theremainder of the molecule. For example, in order to detect either aprotease OR hypoxia (i.e., to cleave X in the presence of eitherprotease or hypoxia), a linker X is designed to place theprotease-sensitive and reduction-sensitive sites in tandem, so thatcleavage of either would suffice to allow separation of the acidicportion A. Alternatively, in order to detect the presence of both aprotease AND hypoxia (i.e., to cleave X in the presence of both proteaseand hypoxia but not in the presence of only one alone), a linker X isdesigned to place the protease sensitive site between at least one pairof cysteines that are disulfide-bonded to each other. In that case, bothprotease cleavage and disulfide reduction are required in order to allowseparation of portion A.

Macromolecular Carrier

Disclosed herein, in certain embodiments, is a selective transportmolecule with increased in vivo circulation. In some embodiments, aselective transport molecule disclosed herein has the formula(A-X-B-C)_(n)-M, wherein C is a cargo moiety; A is a peptide with asequence comprising 5 to 9 consecutive acidic amino acids, wherein theamino acids are selected from: aspartates and glutamates; B is a peptidewith a sequence comprising 5 to 20 consecutive basic amino acids; X is alinker; M is a macromolecular carrier; and n is an integer between 1 and20.

The term “carrier” means an inert molecule that increases (a) plasmahalf-life and (b) solubility.

In some embodiments, a carrier decreases uptake of a selective transportmolecule into cartilage. In some embodiments, a carrier decreases uptakeof a selective transport molecule into joints. In some embodiments, acarrier decreases uptake of a selective transport molecule into theliver. In some embodiments, a carrier decreases uptake of a selectivetransport molecule into kidneys.

In some embodiments, a carrier increases plasma half-life and solubilityby reducing glomerular filtration. In some embodiments, a carrierincreases tumor uptake due to enhanced permeability and retention (EPR)of tumor vasculature.

In some embodiments, M is bound to A. In some embodiments, M is bound toA at the n-terminal poly glutamate. In some embodiments, M is bound to A(or, the n-terminal poly glutamate) by a covalent linkage. In someembodiments, the covalent linkage comprises an ether bond, thioetherbond, amine bond, amide bond, carbon-carbon bond, carbon-nitrogen bond,carbon-oxygen bond, or carbon-sulfur bond.

In some embodiments, M is bound to B. In some embodiments, M is bound toB at the c-terminal polyarginine. In some embodiments, M is bound to B(or, the c-terminal polyarginine) by a covalent linkage. In someembodiments, the covalent linkage comprises an ether bond, thioetherbond, amine bond, amide bond, carbon-carbon bond, carbon-nitrogen bond,carbon-oxygen bond, or carbon-sulfur bond.

In some embodiments, M is selected from a protein, a synthetic ornatural polymer, or a dendrimer. In some embodiments, M is selected fromdextran, a PEG polymer (e.g., PEG 5 kDa and PEG 121cDa), albumin, or acombination thereof. In some embodiments, M is a PEG polymer.

In some embodiments, the size of the carrier is between 50 and 70kD. Insome embodiments, small amounts of negative charge keep peptides out ofthe liver while not causing synovial uptake.

In some embodiments, the selective transport molecule is conjugated toalbumin. In certain instances, albumin is excluded from the glomerularfiltrate under normal physiological conditions. In some embodiments, theselective transport molecule comprises a reactive group such asmaleimide that can form a covalent conjugate with albumin. A selectivetransport molecule comprising albumin results in enhanced accumulationof cleaved selective transport molecules in tumors in a cleavagedependent manner. See, Example 2. In some embodiments, albuminconjugates have good pharmacokinetic properties but are difficult towork with synthetically.

In some embodiments, the selective transport molecule is conjugated to aPEG polymer. In some embodiments, the selective transport molecule isconjugated to a PEG 5 kDa polymer. In some embodiments, the selectivetransport molecule is conjugated to a PEG 121(Da polymer. In someembodiments, 5kD PEG conjugates behaved similarly to free peptides. Insome embodiments, 121cD PEG conjugates had a longer halflife as comparedto free peptides. See Example 5 for a detailed analysis of the effectsof using a PEG polymer.

In some embodiments, the selective transport molecule is conjugated to adextran. In some embodiments, the selective transport molecule isconjugated to a 70 kDa dextran. In some embodiments, dextran conjugates,being a mixture of molecular weights, are difficult to synthesize andpurify reproducibly. See Example 5 for a detailed analysis of theeffects of using a dextran.

In some embodiments, the selective transport molecule is conjugated tostreptavidin. See Example 5 for a detailed analysis of the effects ofusing streptavadin.

In some embodiments, the selective transport molecule is conjugated to afifth generation PAMAM dendrimer.

In some embodiments, a carrier is capped. See Example 1 for methods ofcapping. In some embodiments, capping a carrier improves thepharmacokinetics and reduces cytotoxicity of a carrier by addinghydrophilicity. In some embodiments, the cap is selected from: Acetyl,succinyl, 3-hydroxypropionyl, 2-sulfobenzoyl, glycidyl, PEG-2, PEG-4,PEG-8 and PEG-12. For a detailed analysis of the effects of capping, seeExample 6.

Dendrimers

Disclosed herein, in certain embodiments, is a selective transportmolecule. In some embodiments, a selective transport molecule disclosedherein has the formula (A-X-B)_(n)-D, wherein D is a dendrimer; A is apeptide with a sequence comprising 5 to 9 consecutive acidic aminoacids, wherein the amino acids are selected from: aspartates andglutamates; B is a peptide with a sequence comprising 5 to 20consecutive basic amino acids; X is a linker; and n is an integerbetween 1 and 20; and wherein D is bound to an (A-X-B) moiety by a bondwith a B. In some embodiments, D is bound to an (A-X-B) moiety by a bondwith a polyarginine terminus. In some embodiments, D comprises at leastone cargo moiety. See Example 1 for method of conjugating a peptide to adendrimer.

As used herein, “dendrimer” means a poly-functional (or, poly-branched)molecule. In some embodiments, a denrimer is a structure in which acentral molecule branches repetitively and repetitiously. In someembodiments, the dendrimer is a nanoparticle.

In some embodiments, D is bound to B (or, the c-terminal polyarginine)by a covalent linkage. In some embodiments, the covalent linkagecomprises an ether bond, thioether bond, amine bond, amide bond,carbon-carbon bond, carbon-nitrogen bond, carbon-oxygen bond, orcarbon-sulfur bond.

In some embodiments, a plurality of (A-X-B) moieties are attached to D.See, Example 3. In some embodiments, a plurality of cargo moieties areattached to D. In some embodiments, (a) a plurality of (A-X-B) moietiesare attached to D; and (b) a plurality of cargo moieties are attached toD.

In some embodiments, the dendrimer comprises a reactive group such asmaleimide that can form a covalent conjugate with albumin. In someembodiments, a dendrimer is conjugated to a selective transport moleculevia a maleimide linker at the C-terminal end of the selective transportmolecule.

In some embodiments, conjugating a selective transport molecule to adendrimer increases plasma half-life as compared to an unconjugated (or,free) selective transport molecule. In some embodiments, a selectivetransport molecule conjugated to a dendrimer results in a decrease inacute toxicity as compared to unconjugated selective transportmolecules. In some embodiments, a selective transport moleculeconjugated to a dendrimer reduces uptake by synovium, cartilage andkidney as compared to unconjugated selective transport molecules.

In some embodiments, a selective transport molecule conjugated to adendrimeric nanoparticle is used to target tumor associated macrophages.In some embodiments, a selective transport molecule conjugated to adendrimeric nanoparticle, wherein the nanoparticle further comprisesRicin A, is used to poison subcutaneous macrophages. See Example 7 for adetailed analysis of the effects of using a dendrimeric nanoparticle.

Cargo

Disclosed herein, in certain embodiments, is a selective transportmolecule.

In some embodiments, a selective transport molecule disclosed herein hasthe formula A-X-B-C, wherein C is a cargo moiety; A is a peptide with asequence comprising 5 to 9 consecutive acidic amino acids, wherein theamino acids are selected from: aspartates and glutamates; B is a peptidewith a sequence comprising 5 to 20 consecutive basic amino acids; and Xis a linker that is cleavable by thrombin.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B-C)_(n)-M, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; M is amacromolecular carrier; and n is an integer between 1 and 20.

In some embodiments, a selective transport molecule disclosed herein hasthe formula (A-X-B)_(n)-D, wherein C is a cargo moiety; A is a peptidewith a sequence comprising 5 to 9 consecutive acidic amino acids,wherein the amino acids are selected from: aspartates and glutamates; Bis a peptide with a sequence comprising 5 to 20 consecutive basic aminoacids; X is a linker that is cleavable by thrombin; D is a dendrimer;and n is an integer between 1 and 20. In some embodiments, D comprises acargo moiety. See Example 1 for methods of attaching a label to aselective transport molecule.

Selective transport molecules disclosed herein are suitable for carryingdifferent cargoes, including different types of cargoes and differentspecies of the same types of cargo, for uptake into cells. For example,different types of cargo may include marker cargoes (e.g., fluorescentor radioactive label moieties) and therapeutic cargoes (e.g.,chemotherapeutic molecules such as methotrexate or doxorubicin), orother cargoes. Where destruction of aberrant or diseased cells istherapeutically required, a therapeutic cargo may include a “cytotoxicagent,” i.e., a substance that inhibits or prevents the function ofcells and/or causes destruction of cells.

Delivery of cargo such as a fluorescent molecule may be used tovisualize cells having a certain condition or cells in a regionexhibiting a particular condition. For example, thrombosis (clotformation) may be visualized by designing a linker X to be cleaved bythrombin. Thus, fluorescent molecules are one example of a marker thatmay be delivered to target cells and regions upon release of a portion Aupon cleavage of a linker X.

In some embodiments, the cargo moiety is selected from an imaging agent,a therapeutic agent, a lipid, or a combination thereof.

In some embodiments, the cargo portion comprises at least two cargomoieties. In some embodiments, C comprises a marker cargo and atherapeutic cargo. Multiple cargo moieties may allow, for example,delivery of both a radioactive marker and an ultrasound or contrastagent, allowing imaging by different modalities. Alternatively, forexample, delivery of radioactive cargo along with an anti-cancer agent,providing enhanced anticancer activity, or delivery of a radioactivecargo with a fluorescent cargo, allowing multiple means of localizingand identifying cells which have taken up cargo.

Attachment of a Cargo Moiety

The cargo moiety is attached to B in any location or orientation. Thecargo moiety need not be located at an opposite end of portion B than alinker X. Any location of attachment of the cargo moiety to B isacceptable as long as that attachment remains after X is cleaved. Forexample, the cargo moiety may be attached to or near to an end ofportion B with linker X attached to an opposite end of portion B asillustrated in FIGS. 2A and 2B. The cargo moiety may also be attached toor near to an end of portion B with linker X attached to or near to thesame end of portion B.

Wherein the molecule comprises a dendrimer, the cargo is attacheddirectly to D. By way of non-limiting example, the cargo is attached asfollows:

-   -   (A-X-B)_(n)-D-cargo.        Imaging Agents as Cargo

In some embodiments, a cargo moiety is a fluorescent molecule such asfluorescein. Fluorescent cargo moieties enable easy measurement byfluorescence microscopy or flow cytometry in unfixed cultured cells.

In some embodiments, a cargo moiety is labeled with a positron-emittingisotope (e.g., ¹⁸F) for positron emission tomography (PET), gamma-rayisotope (e.g., ^(99m)Tc) for single photon emission computed tomography(SPECT), a paramagnetic molecule or nanoparticle (e.g., G³⁺ chelate orcoated magnetite nanoparticle) for magnetic resonance imaging (MRI), anear-infrared fluorophore for near-infra red (near-IR) imaging, aluciferase (firefly, bacterial, or coelenterate) or other luminescentmolecule for bioluminescence imaging, or a perfluorocarbon-filledvesicle for ultrasound.

In some embodiments, a cargo moiety is a radioactive moiety, for examplea radioactive isotope such as ²¹¹At, ¹³¹I, ¹²⁵I, ⁹⁰Y, ¹⁸⁶Re, ¹⁸⁸Re,¹⁵³Sm, ²¹²Bi, ³²P, radioactive isotopes of Lu, and others.

In some embodiments, a cargo moiety is a fluorescent moiety, such as afluorescent protein, peptide, or fluorescent dye molecule. Commonclasses of fluorescent dyes include, but are not limited to, xanthenessuch as rhodamines, rhodols and fluoresceins, and their derivatives;bimanes; coumarins and their derivatives such as umbelliferone andaminomethyl coumarins; aromatic amines such as dansyl; squarate dyes;benzofurans; fluorescent cyanines; carbazoles; dicyanomethylene pyranes,polymethine, oxabenzanthrane, xanthene, pyrylium, carbostyl, perylene,acridone, quinacridone, rubrene, anthracene, coronene, phenanthrecene,pyrene, butadiene, stilbene, lanthanide metal chelate complexes,rare-earth metal chelate complexes, and derivatives of such dyes.Fluorescent dyes are discussed, for example, in U.S. Pat. No. 4,452,720,U.S. Pat. No. 5,227,487, and U.S. Pat. No. 5,543,295.

In some embodiments, a cargo moiety is a fluorescein dye. Typicalfluorescein dyes include, but are not limited to, 5-carboxyfluorescein,fluorescein-5-isothiocyanate and 6-carboxyfluorescein; examples of otherfluorescein dyes can be found, for example, in U.S. Pat. No. 6,008,379,U.S. Pat. No. 5,750,409, U.S. Pat. No. 5,066,580, and U.S. Pat. No.4,439,356. A cargo moiety C may include a rhodamine dye, such as, forexample, tetramethylrhodamine-6-isothiocyanate,5-carboxytetramethylrhodamine, 5-carboxy rhodol derivatives, tetramethyland tetraethyl rhodamine, diphenyldimethyl and diphenyldiethylrhodamine, dinaphthyl rhodamine, rhodamine 101 sulfonyl chloride (soldunder the tradename of TEXAS REDO), and other rhodamine dyes. Otherrhodamine dyes can be found, for example, in U.S. Pat. No. 6,080,852,U.S. Pat. No. 6,025,505, U.S. Pat. No. 5,936,087, U.S. Pat. No.5,750,409. A cargo moiety C may include a cyanine dye, such as, forexample, Cy3, Cy3B, Cy3.5, Cy5, Cy5.5, Cy 7.

Some of the above compounds or their derivatives will producephosphorescence in addition to fluorescence, or will only phosphoresce.Some phosphorescent compounds include porphyrins, phthalocyanines,polyaromatic compounds such as pyrenes, anthracenes and acenaphthenes,and so forth, and may be, or may be included in, a cargo moiety. A cargomoiety may also be or include a fluorescence quencher, such as, forexample, a (4-dimethylamino-phenylazo)benzoic acid (DABCYL) group.

In some embodiments, a cargo moiety is a fluorescent label. In someembodiments, a cargo moiety is indocarbocyanine dye, Cy5, Cy5.5, Cy7,IR800CW, or a combination thereof. In some embodiments, a cargo moietyis a MRI contrast agent. In some embodiments, a cargo moiety is Gdcomplex of[4,7,10-tris(carboxymethyl)-1,4,7,10-tetraazacyclododec-1-yl]acetyl.

In some embodiments, a cargo moiety is all or part of a molecularbeacon. As used herein, “molecular beacon” means a pair of connectedcompounds having complementary regions with a fluorophore and afluorescent quencher so that the fluorescence of the fluorophore isquenched by the quencher. One or both of the complementary regions maybe part of the cargo moiety. Where only one of the complementary regions(e.g., the fluorescent moiety) is part of the cargo moiety, and wherethe quencher moiety is part of the linker X or the acidic portion A,then cleavage of the linker X will allow fluorescence of the fluorescentportion and detection of the cleavage. Upon cellular uptake, thefluorescent portion of a molecular beacon will allow detection of thecell. For example, a quencher Q may be attached to an acidic portion Ato form a selective transport molecule having features of the inventionQ-A-X-B-C where cargo is fluorescent and is quenched by Q. The quenchingof the cargo moiety by Q is relieved upon cleavage of X, allowingfluorescent marking of a cell taking up portion B-C. The combination offluorescence dequenching and selective uptake should increase contrastbetween tissues able to cleave X compared to those that cannot cleave X.

Therapeutic Agents as Cargo

In some embodiments, a cargo moiety is a therapeutic agent, such as achemical compound useful in the treatment of cancer, ischemic tissue, ornecrotic tissue.

For therapeutic purposes, for example, suitable classes of cargo includebut are not limited to: a) chemotherapeutic agents; b) radiationsensitizing agents; or c) peptides or proteins that modulate apoptosis,the cell cycle, or other crucial signaling cascades.

In some embodiments, a cargo moiety is an agent that treats acardiovascular disorder. In some embodiments, the cargo moiety is aniacin, a fibrate, a statin, an Apo-A1 mimetic peptide, an apoA-Itransciptional up-regulator, an ACAT inhibitor, a CETP modulator, or acombination thereof, a Glycoprotein (GP) IIb/IIIa receptor antagonist, aP2Y12 receptor antagonist, a Lp-PLA2-inhibitor, a leukotriene inhibitor,a MIF antagonist, or a combination thereof. In some embodiments thecargo moiety is atorvastatin; cerivastatin; fluvastatin; lovastatin;mevastatin; pitavastatin; pravastatin; rosuvastatin; simvastatin;simvastatin and ezetimibe; lovastatin and niacin, extended-release;atorvastatin and amlodipine besylate; simvastatin and niacin,extended-release; bezafibrate; ciprofibrate; clofibrate; gemfibrozil;fenofibrate; DF4 (Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (SEQ ID NO:3)); DFS; RVX-208 (Resverlogix); avasimibe; pactimibe sulfate (CS-505);CI-1011 (2,6-diisopropylphenyl[(2,4,6-triisopropylphenyl)acetyl]sulfamate); CI-976(2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); VULM1457(1-(2,6-diisopropyl-phenyl)-3-[4-(4′-nitrophenylthio)phenyl] urea);CI-976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); E-5324(n-butyl-N′-(2-(3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy)-6-methylphenyl)urea);HL-004 (N-(2,6-diisopropylphenyl) tetradecylthioacetamide); KY-455(N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide); FY-087(N42-[N′-pentyl-(6,6-dimethyl-2,4-heptadiynyl)amino]ethyl]-(2-methyl-1-naphthyl-thio)acetamide);MCC-147 (Mitsubishi Pharma); F 12511((S)-2′,3′,5′-trimethyl-4′-hydroxy-alpha-dodecylthioacetanilide);SMP-500 (Sumitomo Pharmaceuticals); CL 277082(2,4-difluoro-phenyl-NR4-(2,2-dimethylpropyl)phenyl]methyl]-N-(hepthyl)urea);F-1394((1s,2s)-2-[3-(2,2-dimethylpropyl)-3-nonylureido]aminocyclohexane-1-yl3-[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonyl)amino]propionate);CP-113818(N-(2,4-bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic acidamide); YM-750; torcetrapib; anacetrapid; JTT-705 (Japan Tobacco/Roche);abciximab; eptifibatide; tirofiban; roxifiban; variabilin; XV 459(N(3)-(2-(3-(4-formamidinophenyl)isoxazolin-5-yl)acetyl)-N(2)-(1-butyloxycarbonyl)-2,3-diaminopropionate);SR 121566A(3-[N-{4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl1-N-(1-carboxymethylpiperid-4-yl)amino] propionic acid, trihydrochloride); FK419((S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl) propionyl]piperidin-3-ylcarbonyl] amino] propionic acid trihydrate); clopidogrel;prasugrel; cangrelor; AZD6140 (AstraZeneca); MRS 2395(2,2-Dimethyl-propionic acid3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester); BX 667 (Berlex Biosciences); BX 048 (Berlex Biosciences);darapladib (SB 480848); SB-435495 (GlaxoSmithKline); SB-222657(GlaxoSmithKline); SB-253514 (GlaxoSmithKline); A-81834(3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehydeoxime-O-2-acetic acid; AME103 (Amira); AME803 (Amira); atreleuton;BAY-x-1005((R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid);CJ-13610(4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylicacid amide); DG-031 (DeCode); DG-051 (DeCode); MK886(1-[(4-chlorophenyl)methyl]3-[(1,1-dimethylethypthio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoicacid, sodium salt); MK591(3-(1-4[(4-chlorophenyl)methyl]-3-Rt-butylthio)-5-((2-quinoly)methoxy)-1H-indole-2]-,dimehtylpropanoic acid); RP64966([4-[5-(3-Phenyl-propyl)thiophen-2-yl]butoxy] acetic acid); SA6541((R)-S-[[4-(dimethylamino)phenyl]methyl]-N-(3-mercapto-2methyl-1-oxopropyl-L-cycteine);SC-56938 (ethyl-1-[2-[4-(phenylmethyl)phenoxy]ethyl]-4-piperidine-carboxylate); VIA-2291 (Via Pharmaceuticals);WY-47,288 (2-[(1-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138(6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone);or combinations thereof

In some embodiments, the drug is an agent that modulates death (e.g.,via apoptosis or necrosis) of a cell. In some embodiments, the drug is acytotoxic agent. In some embodiments, the drug is maytansine,methotrexate (RHEUMATREX®, Amethopterin); cyclophosphamide (CYTOXAN®);thalidomide (THALIDOMID®); paclitaxel; pemetrexed; pentostatin;pipobroman; pixantrone; plicamycin; procarbazine; proteasome inhibitors(e.g.; bortezomib); raltitrexed; rebeccamycin; rubitecan; SN-38;salinosporamide A; satraplatin; streptozotocin; swainsonine; tariquidar;taxane; tegafur-uracil; temozolomide; testolactone; thioTEPA;tioguanine; topotecan; trabectedin; tretinoin; triplatin tetranitrate;tris(2-chloroethyl)amine; troxacitabine; uracil mustard; valrubicin;vinbiastine; vincristine; vinorelbine; vorinostat; zosuquidar; or acombination thereof. In some embodiments, the drug is a pro-apoptoticagent. In some embodiments, the drug is an anti-apoptotic agent. In someembodiments, the drug is selected from: minocycline; SB-203580(4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)1H-imidazole); PD 169316(4-(4-Fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole); SB202190(4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole); RWJ67657(4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol);SB 220025(542-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinlypimidazole);D-JNKI-1 ((D)-hJIP175-157-DPro-DPro-(D)-HIV-TAT57-48); AM-111 (Auris);SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one); JNK Inhibitor I((L)-HIV-TAT48-57-PP-JBD20); JNK Inhibitor III((L)-HIV-TAT47-57-gaba-c-Jun533-57); AS601245 (1,3-benzothiazol-2-yl(2-[[2-(3-pyridinyl) ethyl] amino]-4 pyrimidinyl) acetonitrile); JNKInhibitor VI (H2N-RPKRPTTLNLF-NH2 (SEQ ID NO: 7)); JNK Inhibitor VIII(N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxyphenyl)acetamide);JNK Inhibitor IX(N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)-1-naphthamide);dicumarol (3,3′-Methylenebis(4-hydroxycoumarin)); SC-236(4-[5-(4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzene-sulfonamide);CEP-1347 (Cephalon); CEP-11004 (Cephalon); an artificial proteincomprising at least a portion of a Bcl-2 polypeptide; a recombinant FNK;V5 (also known as Bax inhibitor peptide V5); Bax channel blocker((±)-1-(3,6-Dibromocarbazol-9-yl)-3-piperazin-1-yl-propan-2-01); Baxinhibiting peptide P5 (also known as Bax inhibitor peptide P5); Kp7-6;FAIM(S) (Fas apoptosis inhibitory molecule-short); FAIM(L) (Fasapoptosis inhibitory molecule-long); Fas:Fc; FAP-1; NOK2; F2051; F1926;F2928; ZB4; Fas M3 mAb; EGF; 740 Y-P; SC 3036 (KKHTDDGYMPMSPGVA (SEQ IDNO: 8)); PI 3-kinase Activator (Santa Cruz Biotechnology, Inc.); Pam3Cys((S)-(2,3-bis(palmitoyloxy)-(2R5)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-0H,trihydrochloride); Act1 (NF-kB activator 1); an anti-IkB antibody;Acetyl-11-keto-b-Boswellic Acid; Andrographolide; Caffeic Acid PhenethylEster (CAPE); Gliotoxin; Isohelenin; NEMO-Binding Domain Binding Peptide(DRQIKIWFQNRRMKWKKTALDWSWLQTE (SEQ ID NO: 9)); NF-kB ActivationInhibitor (6-Amino-4-(4-phenoxyphenylethylamino)quinazoline); NF-kBActivation Inhibitor H(4-Methyl-N1-(3-phenylpropyl)benzene-1,2-diamine); NF-kB ActivationInhibitor III (3-Chloro-4-nitro-N-(5-nitro-2-thiazolyl)-benzamide);NF-kB Activation Inhibitor IV ((E)-2-Fluoro-4′-methoxystilbene); NF-kBActivation Inhibitor V(5-Hydroxy-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione); NF-kB SN50(AAVALLPAVLLALLAPVQRKRQKLMP (SEQ ID NO: 10)); Oridonin; Parthenolide;PPM-18 (2-Benzoylamino-1,4-naphthoquinone); Ro106-9920; Sulfasalazine;TIRAP Inhibitor Peptide (RQIKIWFNRRMKWKKLQLRDAAPGGAIVS (SEQ ID NO: 11));Withaferin A; Wogonin; BAY 11-7082((E)34(4-Methylphenyl)sulfonyl]-2-propenenitrile); BAY 11-7085((E)3-[(4-t-Butylphenyl)sulfonyl]-2-propenenitrile); (E)-Capsaicin;Aurothiomalate (ATM or AuTM); Evodiamine; Hypoestoxide; IKK InhibitorIII (BMS-345541); IKK Inhibitor VII; IKK Inhibitor X; IKK Inhibitor II;IKK-2 Inhibitor IV; IKK-2 Inhibitor V; IKK-2 Inhibitor VI; IKK-2Inhibitor (SC-514); IkB Kinase Inhibitor Peptide; IKK-3 Inhibitor IX;ARRY-797 (Array BioPharma); SB-220025(5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinly)imidazole);SB-239063(trans-4-[4-(4-Fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol);SB-202190(4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole);JX-401-[2-Methoxy-4-(methylthio)benzoyl]-4-(phenylmethyl)piperidine);PD-169316(4-(4-Fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole);SKF-86002 (6-(4-Fluorophenyl)-2,3-dihydro-5-(4-pyridinyl)imidazo[2,1-b]thiazole dihydrochloride); SB-200646(N-(1-Methyl-1H-indol-5-yl)-N-3-pyridinylurea); CMPD-1(2′-Fluoro-N-(4-hydroxyphenyl)-[1,1′-biphenyl]-4-butanamide); EO-1428((2-Methylphenyl)-[4-[(2-amino-4-bromophenyl)amino]-2-chlorophenyl]methanone);SB-253080(4-[5-(4-Fluorophenyl)-2-[4-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine);SD-169 (1H-Indole-5-carboxamide); SB-203580(4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)1H-imidazole); TZP-101 (Tranzyme Pharma); TZP-102 (Tranzyme Pharma);GHRP-6 (growth hormone-releasing peptide-6); GHRP-2 (growthhormone-releasing peptide-2); EX-1314 (Elixir Pharmaceuticals); MK-677(Merck); L-692,429 (Butanamide,3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-((2′-(1H-tetrazol-5-yl)(1,1′-biphenyl)-4-yl)methyl)-1H-1-benzazepin-3-yl)-,(R)-); EP1572 (Aib-DTrp-DgTrp-CHO); diltiazem; metabolites of diltiazem;BRE (Brain and Reproductive organ-Expressed protein); verapamil;nimodipine; diltiazem; omega-conotoxin; GVIA; amlodipine; felodipine;lacidipine; mibefradil; NPPB (5-Nitro-2-(3-phenylpropylamino)benzoicAcid); flunarizine; erythropoietin; piperine; hemin; brazilin; z-VAD-FMK(Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone); z-LEHD-FMK(benzyloxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-fluoromethylketone) (SEQ IDNO: 12); B-D-FMK (boc-aspartyl(Ome)-fluoromethylketone); Ac-LEHD-CHO(N-acetyl-Leu-Glu-His-Asp-CHO) (SEQ ID NO: 13); Ac-IETD-CHO(N-acetyl-Ile-Glu-Thr-Asp-CHO) (SEQ ID NO: 14); z-IETD-FMK(benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethy lketone) (SEQID NO: 15); FAM-LEHD-FMK (benzyloxycarbonyl Leu-Glu-His-Asp-fluoromethylketone) (SEQ ID NO: 16); FAM-LETD-FMK (benzyloxycarbonylLeu-Glu-Thr-Asp-fluoromethyl ketone) (SEQ ID NO: 17); Q-VD-OPH(Quinoline-Val-Asp-CH2-O-Ph); XIAP; cIAP-1; cIAP-2; ML-IAP; ILP-2; NAIP;Survivin; Bruce; IAPL-3; fortilin; leupeptine; PD-150606(3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid); MDL-28170(Z-Val-Phe-CHO); calpeptin; acetyl-calpastatin; MG 132(N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide);MYODUR; BN 82270 (Ipsen); BN 2204 (Ipsen); AHLi-11 (QuarkPharmaceuticals), an mdm2 protein, pifithrin-a(1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanone);trans-stilbene; cis-stilbene; resveratrol; piceatannol; rhapontin;deoxyrhapontin; butein; chalcon; isoliquirtigen; butein;4,2′,4′-trihydroxychalcone; 3,4,2′,4′,6′-pentahydroxychalcone; flavone;morin; fisetin; luteolin; quercetin; kaempferol; apigenin; gossypetin;myricetin; 6-hydroxyapigenin; 5-hydroxyflavone;5,7,3′,4′,5′-pentahydroxyflavone; 3,7,3′,4′,5′-pentahydroxyflavone;3,6,3′,4′-tetrahydroxyflavone; 7,3′,4′,5′-tetrahydroxyflavone;3,6,2′,4′-tetrahydroxyflavone; 7,4′-dihydroxyflavone;7,8,3′,4′-tetrahydroxyflavone; 3,6,2′,3′-tetrahydroxyflavone;4′-hydroxyflavone; 5-hydroxyflavone; 5,4′-dihydroxyflavone;5,7-dihydroxyflavone; daidzein; genistein; naringenin; flavanone;3,5,7,3′,4′-pentahydroxyflavanone; pelargonidin chloride; cyanidinchloride; delphinidin chloride; (−)-epicatechin (Hydroxy Sites:3,5,7,3′,4′); (−)-catechin (Hydroxy Sites: 3,5,7,3′,4′);(−)-gallocatechin (Hydroxy Sites: 3,5,7,3′,4′,5′) (+)-catechin (HydroxySites: 3,5,7,3′,4′); (+)-epicatechin (Hydroxy Sites: 3,5,7,3′,4′);Hinokitiol (b-Thujaplicin;2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one); L-(+)-Ergothioneine((S)-a-Carboxy-2,3-dihydro-N,N,N-trimethyl-2-thioxo-1H-imidazole4-ethanaminiuminner salt); Caffeic Acid Phenyl Ester; MCI-186(3-Methyl-1-phenyl-2-pyrazolin-5-one); HBED(N,N′-Di-(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid.H2O;Ambroxol (trans-4-(2-Amino-3,5-dibromobenzylamino)cyclohexane-HCl andU83836E((-2-((4-(2,6-di-1-Pyrrolidinyl-4-pyrimidinyl)-1-piperzainyl)methyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol.2HCl;β-1′-5-methyl-nicotinamide-2′-deoxyribose;β-D-1′-5-methyl-nicotinamide-2′-deoxyribofuranoside; (β-1′-4,5 dimethylnicotinamide-2′-de-oxyribose;β-D-1′-4,5-dimethyl-nicotinamide-2′-deoxyribofuranoside; 1-Naphthyl PP1(1-(1,1-Dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine); Lavendustin A(5-[[(2,5-Dihydroxyphenyl)methyl][(2-hydroxyphenyl)methyl]amino]-2-hydroxybenzoicacid); MNS (3,4-Methylenedioxy-h-nitrostyrene); PP1(1-(1,1-Dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo [3,4-d]pyrimidin-4-amine); PP2 (3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine); KX-004(Kinex); KX-005 (Kinex); KX-136 (Kinex); KX-174 (Kinex); KX-141 (Kinex);KX2-328 (Kinex); KX-306 (Kinex); KX-329 (Kinex); KX2-391 (Kinex);KX2-377 (Kinex); ZD4190 (Astra Zeneca;N-(4-bromo-2-fluorophenyl)-6-methoxy-7-(2-(1H-1,2,3-triazol-1-yl)ethoxy)quinazolin-4-amine);AP22408 (Ariad Pharmaceuticals); AP23236 (Ariad Pharmaceuticals);AP23451 (Ariad Pharmaceuticals); AP23464 (Ariad Pharmaceuticals);AZD0530 (Astra Zeneca); AZM475271 (M475271; Astra Zeneca); Dasatinib(N-(2-chloro-6-methylphneyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide); GN963(trans-4-(6,7-dimethoxyquinoxalin-2ylamino)cyclohexanol sulfate);Bosutinib(4-((2,4-dichloro-5-methoxyphenyl)amino)-6-methoxy-7-(3-(4-methyl-1-piperazinyl)propoxy)-3-quinolinecarbonitrile);or combinations thereof.

Lipids as Cargo

Disclosed herein, in certain embodiments, is a selective transportmolecule. In some embodiments, a selective transport molecule disclosedherein has the formula (A-X-B)_(n)-L, wherein L is a lipid; A is apeptide with a sequence comprising 5 to 9 consecutive acidic aminoacids, wherein the amino acids are selected from: aspartates andglutamates; B is a peptide with a sequence comprising 5 to 20consecutive basic amino acids; X is a linker; and n is an integerbetween 1 and 20; and wherein L is bound to an (A-X-B) moiety by a bondwith a B.

In some embodiments, the lipid entraps a hydrophobic molecule. In someembodiments, the lipid entraps at least one agent selected from thegroup consisting of a therapeutic moiety or an imaging moiety.

In some embodiments, the lipid is PEGylated. In some embodiments, thelipid is PEG(2K)-phosphatidylethanolamine.

Methods of Use

Visualizing Thrombin

Disclosed herein, in certain embodiments, is a method of imagingthrombin activity in a subject. In some embodiments, the methodcomprises imaging thrombin activity after the subject has beenadministered a selective transport molecule disclosed herein.

In some embodiments, an increase in thrombin activity over normal rangesindicates the presence of a cancer, ischemia, or an atheroscleroticplaque.

In some embodiments, the signal intensity of an imaging agentcorresponds to the total atherosclerotic plaque burden, histologic stageof the atherosclerotic plaque, and provides evidence of recent plaquerupture. In some embodiments, administering a selective transportmolecule disclosed herein allows a medical professional to image thesurgical margins of an atherosclerotic plaque for removal.

In some embodiments, the signal intensity of an imaging agentcorresponds to the size of the tumor. In some embodiments, administeringa selective transport molecule disclosed herein allows a medicalprofessional to evaluate the progression or regression of a tumor. Insome embodiments, administering a selective transport molecule disclosedherein allows a medical professional to image the surgical margins for atumor or tissue resection in a subject in need thereof

In some embodiments, the signal intensity of an imaging agentcorresponds to amount of ischemia and the damage to the surroundingcells. As used herein, “ischemia” means a shortage of the blood supplyto an organ, (i.e. a shortage of oxygen, glucose and other blood-bornefuels). In some embodiments, ischemia is caused by occlusion of a vesselor artery (e.g., due to an embolism or thrombosis). In some embodiments,ischemia is caused by hemorrhage. In some embodiments, ischemia resultsin a stroke. In some embodiments, administering a selective transportmolecule disclosed herein allows a medical professional to evaluate asubject's risk of developing a stroke.

In some embodiments, the image of thrombin activity is memorialized(i.e., a record is created) in print (e.g., as a photograph).

In some embodiments, image of thrombin activity is stored in a computermodule. In some embodiments, the image of thrombin activity is stored incomputer memory. In some embodiments, the image of thrombin activity isstored as a visual file (e.g., JPEG, MPEG, MPEG-2, H.264/MPEG-4 AVC, andSMPTE VC-1). In some embodiments, the image of thrombin activity isstored in volatile computer memory. As used herein, “volatile memory”means computer memory that requires electricity to maintain the storedinformation. In some embodiments, the volatile memory is random accessmemory (RAM), dynamic random access memory (DRAM), or static randomaccess memory (SRAM). In some embodiments, the image of thrombinactivity is stored in non-volatile computer memory. As used herein,“non-volatile memory” means computer memory that retains the storedinformation in the absence of electricity (e.g., hard disks, floppydisks, and magnetic tape, or optical discs). In some embodiments, theimage of thrombin activity is stored on an optical disc (e.g., a Blu-Raydisc, DVD, or a CD). In some embodiments, the image of thrombin activityis stored on a magnetic storage device.

Visualizing Tumors

In some embodiments, a selective transport molecule disclosed herein isadministered to a subject in need thereof to visualize a tumor. In someembodiments, a selective transport molecule disclosed herein isadministered to a subject in need thereof prior to surgery. In someembodiments, a selective transport molecule disclosed herein isadministered to a subject in need thereof prior to surgery and used todefine the surgical margins of a tumor. In some embodiments, a dualmodality (MR and fluorescence) selective transport molecule allowspre-operative staging by oncologists and radiologists, particularly forcancers such as prostate where invasion of a capsule is important,preventing surgery on patients who are non-operative candidates. In someembodiments, the anatomical and biochemical information given by thedual label selective transport molecule are useful for surgeons inplanning complex surgical procedures. In some embodiments, tight bindingof selective transport molecules to the site of cleavage provideslocalized information regarding tumor biology that not only allows thesurgeon to focus on the most invasive areas of tumor growth withintraoperative fluorescence imaging but also allows the pathologist todo the same with intraoperative histology. Following surgery, in someembodiments, the dual probe allows further evaluation for completenessof tumor removal with a second MRI.

In some embodiments, the image of a tumor is memorialized (i.e., arecord is created) in print (e.g., as a photograph).

In some embodiments, image of a tumor is stored in a computer module. Insome embodiments, the image of a tumor is stored in computer memory. Insome embodiments, the image of a tumor is stored as a visual file (e.g.,JPEG, MPEG, MPEG-2, H.264/MPEG-4 AVC, and SMPTE VC-1). In someembodiments, the image of a tumor is stored in volatile computer memory.As used herein, “volatile memory” means computer memory that requireselectricity to maintain the stored information. In some embodiments, thevolatile memory is random access memory (RAM), dynamic random accessmemory (DRAM), or static random access memory (SRAM). In someembodiments, the image of a tumor is stored in non-volatile computermemory. As used herein, “non-volatile memory” means computer memory thatretains the stored information in the absence of electricity (e.g., harddisks, floppy disks, and magnetic tape, or optical discs). In someembodiments, the image of a tumor is stored on an optical disc (e.g., aBlu-Ray disc, DVD, or a CD). In some embodiments, the image of a tumoris stored on a magnetic storage device.

Visualizing Ischemia

In some embodiments, a selective transport molecule disclosed herein isadministered to a subject in need thereof to visualize an area ofischemia. In some embodiments, a selective transport molecule disclosedherein is administered to a subject in need thereof prior to surgery. Insome embodiments, a selective transport molecule disclosed herein isadministered to a subject in need thereof prior to surgery and used todefine an area of ischemia.

In some embodiments, the image of an area of ischemia is memorialized(i.e., a record is created) in print (e.g., as a photograph).

In some embodiments, image of an area of ischemia is stored in acomputer module. In some embodiments, the image of an area of ischemiais stored in computer memory. In some embodiments, the image of an areaof ischemia is stored as a visual file (e.g., JPEG, MPEG, MPEG-2,H.264/MPEG-4 AVC, and SMPTE VC-1). In some embodiments, the image of anarea of ischemia is stored in volatile computer memory. As used herein,“volatile memory” means computer memory that requires electricity tomaintain the stored information. In some embodiments, the volatilememory is random access memory (RAM), dynamic random access memory(DRAM), or static random access memory (SRAM). In some embodiments, theimage of an area of ischemia is stored in non-volatile computer memory.As used herein, “non-volatile memory” means computer memory that retainsthe stored information in the absence of electricity (e.g., hard disks,floppy disks, and magnetic tape, or optical discs). In some embodiments,the image of an area of ischemia is stored on an optical disc (e.g., aBlu-Ray disc, DVD, or a CD). In some embodiments, the image of an areaof ischemia is stored on a magnetic storage device.

Visualizing Atherosclerotic Plaques

In some embodiments, a selective transport molecule disclosed herein isadministered to a subject in need thereof prior to surgery. In someembodiments, a selective transport molecule disclosed herein isadministered to a subject in need thereof prior to surgery and used tohighlight high risk plaques intraoperatively. In some embodiments,administering a selective transport molecule disclosed herein prior tosurgery lowers patient morbidity in surgical procedures (e.g., carotidendarterectomy). In some embodiments, administering a selectivetransport molecule disclosed herein prior to surgery lowers patientmorbidity in iatrogenic surgical procedures, coronary artery surgicalprocedures, and mesenteric artery surgical procedures. In someembodiments, administering a selective transport molecule disclosedherein prior to surgery reduces the incidence of embolic events,myocardial infarction and bowel necrosis.

In some embodiments, the image of an atherosclerotic plaque ismemorialized (i.e., a record is created) in print (e.g., as aphotograph).

In some embodiments, image of an atherosclerotic plaque is stored in acomputer module. In some embodiments, the image of an atheroscleroticplaque is stored in computer memory. In some embodiments, the image ofan atherosclerotic plaque is stored as a visual file (e.g., JPEG, MPEG,MPEG-2, H.264/MPEG-4 AVC, and SMPTE VC-1). In some embodiments, theimage of an atherosclerotic plaque is stored in volatile computermemory. As used herein, “volatile memory” means computer memory thatrequires electricity to maintain the stored information. In someembodiments, the volatile memory is random access memory (RAM), dynamicrandom access memory (DRAM), or static random access memory (SRAM). Insome embodiments, the image of an atherosclerotic plaque is stored innon-volatile computer memory. As used herein, “non-volatile memory”means computer memory that retains the stored information in the absenceof electricity (e.g., hard disks, floppy disks, and magnetic tape, oroptical discs). In some embodiments, the image of an atheroscleroticplaque is stored on an optical disc (e.g., a Blu-Ray disc, DVD, or aCD). In some embodiments, the image of an atherosclerotic plaque isstored on a magnetic storage device.

Management of Atherosclerotic Disease

In some embodiments, a selective transport molecule disclosed herein isadministered to a subject to aid in management of atheroscleroticdisease. In some embodiments, a selective transport molecule disclosedherein is administered to a subject to aid a medical professional indistinguishing pathologic features of a plaque with the potential torupture and cause embolic disease. In some embodiments, a selectivetransport molecule disclosed herein is administered to a subject to aida medical professional in distinguishing patients with plaques at highrisk for rupture, or the “high risk patient”. In some embodiments, aselective transport molecule disclosed herein is administered to asubject to aid a medical professional in characterizing the riskassociated with an atherosclerotic plaque in a subject, wherein the riskis proportional to the imaging agent signal intensity. Currently tissueexamination of excised carotid endarterectomy fragments includes lookingfor distinctive pathological features such as fissures,micro-ulcerations, microthrombi or calcified nodules. This task is muchmore difficult in specimens that do not contain vessel wall, leading tosignificant intraobserver variability in identifying the rupturepotential of surgically excised plaques (e.g., those removed duringcarotid endarterectomy).

Drug Delivery

Disclosed herein, in certain embodiments, are methods of targeted drugdelivery. In some embodiments, a selective transport molecule describedherein delivers a drug to a specific target (e.g., a cell or a pluralityof cells). In some embodiments, a selective transport molecule describedherein delivers a drug to a cell or tissue characterized by elevatedthrombin levels when compared to normal cell or tissue conditions.

In some embodiments, the drug is an agent that treats a cardiovasculardisorder. In some embodiments, the drug is a niacin, a fibrate, astatin, an Apo-A1 mimetic peptide, an apoA-I transciptionalup-regulator, an ACAT inhibitor, a CETP modulator, or a combinationthereof, a Glycoprotein (GP) IIb/IIIa receptor antagonist, a P2Y12receptor antagonist, a Lp-PLA2-inhibitor, a leukotriene inhibitor, a MIFantagonist, or a combination thereof. In some embodiments the drug isatorvastatin; cerivastatin; fluvastatin; lovastatin; mevastatin;pitavastatin; pravastatin; rosuvastatin; simvastatin; simvastatin andezetimibe; lovastatin and niacin, extended-release; atorvastatin andamlodipine besylate; simvastatin and niacin, extended-release;bezafibrate; ciprofibrate; clofibrate; gemfibrozil; fenofibrate; DF4(Ac-D-W-F-K-A-F-Y-D-K-V-A-E-K-F-K-E-A-F-NH2 (SEQ ID NO: 3)); DFS;RVX-208 (Resverlogix); avasimibe; pactimibe sulfate (CS-505); CI-1011(2,6-diisopropylphenyl [(2, 4,6-triisopropylphenyl)acetyl]sulfamate);CI-976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); VULM1457(1-(2,6-diisopropyl-phenyl)-3[4-(4′-nitrophenylthio)phenyl] urea);CI-976 (2,2-dimethyl-N-(2,4,6-trimethoxyphenyl)dodecanamide); E-5324(n-butyl-N′-(2-(3-(5-ethyl-4-phenyl-1H-imidazol-1-yl)propoxy)-6-methylphenyl)urea);HL-004 (N-(2,6-diisopropylphenyl) tetradecylthioacetamide); KY-455(N-(4,6-dimethyl-1-pentylindolin-7-yl)-2,2-dimethylpropanamide); FY-087(N[2-[N′-pentyl-(6,6-dimethyl-2,4-heptadiynyl(amino]ethyl]-(2-methyl-1-naphthyl-thio)acetamide);MCC-147 (Mitsubishi Pharma); F 12511((S)-2′,3′,5′-trimethyl-4′-hydroxy-alpha-dodecylthioacetanilide);SMP-500 (Sumitomo Pharmaceuticals); CL 277082(2,4-difluoro-phenyl-N[[4-(2,2-dimethylpropyl)phenylimethyl]-N-(hepthyl(urea);F-1394((1s,2s)-2[3-(2,2-dimethylpropyl)-3-nonylureido]aminocyclohexane-1-yl3[N-(2,2,5,5-tetramethyl-1,3-dioxane-4-carbonylamino]propionate);CP-113818(N-(2,4-bis(methylthio)-6-methylpyridin-3-yl)-2-(hexylthio)decanoic acidamide); YM-750; torcetrapib; anacetrapid; JTT-705 (Japan Tobacco/Roche);abciximab; eptifibatide; tirofiban; roxifiban; variabilin; XV 459(N(3)-(2-(3-(4-formamidinophenyl)isoxazolin-5-yl)acetyl)-N(2)-(1-butyloxycarbonyl)-2,3-diaminopropionate);SR 121566A(3[N-{4-[4-(aminoiminomethyl)phenyl]-1,3-thiazol-2-yl}-N-(1-carboxymethylpiperid-4-yl)aminol propionic acid, trihydrochioride); FK419((S)-2-acetylamino-3-[(R)-[1-[3-(piperidin-4-yl) propionyl]piperidin-3-ylcarbonyl] amino] propionic acid trihydrate); clopidogrel;prasugrel; cangrelor; AZD6140 (AstraZeneca); MRS 2395(2,2-Dimethyl-propionic acid3-(2-chloro-6-methylaminopurin-9-yl)-2-(2,2-dimethyl-propionyloxymethyl)-propylester); BX 667 (Berlex Biosciences); BX 048 (Berlex Biosciences);darapladib (SB 480848); SB-435495 (GlaxoSmithKline); SB-222657(GlaxoSmithKline); SB-253514 (GlaxoSmithKline); A-81834(3-(3-(1,1-dimethylethylthio-5-(quinoline-2-ylmethoxy)-1-(4-chloromethylphenyl)indole-2-yl)-2,2-dimethylpropionaldehydeoxime-O-2-acetic acid; AME103 (Amira); AME803 (Amira); atreleuton;BAY-x-1005((R)-(+)-alpha-cyclopentyl-4-(2-quinolinylmethoxy)-Benzeneacetic acid);CJ-13610(4-(3-(4-(2-Methyl-imidazol-1-yl)-phenylsulfanyl)-phenyl)-tetrahydro-pyran-4-carboxylicacid amide); DG-031 (DeCode); DG-051 (DeCode); MK886 (1[(4-chlorophenyl)methyl]3-[(1,1-dimethylethyl)thio]-α,α-dimethyl-5-(1-methylethyl)-1H-indole-2-propanoicacid, sodium salt); MK591(3-(1-4[(4-chlorophenyl)methyl]-3-[(t-butylthio)-5-((2-quinoly)methoxy)-1H-indole-2]-,dimehtylpropanoic acid); RP64966([4-[5-(3-Phenyl-propyl)thiophen-2-yl]butoxy] acetic acid); SA6541((R)-S-[[4-(dimethylamino)phenyl]methyl]-N-(3-mercapto-2methyl-1-oxopropyl-L-cycteine);SC-56938 (ethyl-1-[2-[4-(phenylmethyl)phenoxy]ethyl]-4-piperidine-carboxylate); VIA-2291 (Via Pharmaceuticals);WY-47,288 (2-[(1-naphthalenyloxy)methyl]quinoline); zileuton; ZD-2138(6-((3-fluoro-5-(tetrahydro-4-methoxy-2H-pyran-4yl)phenoxy)methyl)-1-methyl-2(1H)-quinlolinone);or combinations thereof

In some embodiments, the drug is an agent that modulates death (e.g.,via apoptosis or necrosis) of a cell. In some embodiments, the drug is acytotoxic agent. In some embodiments, the drug is maytansine,methotrexate (RHEUMATREX®, Amethopterin); cyclophosphamide(CYTOXAN®);thalidomide (THALIDOMID®); paclitaxel; pemetrexed;pentostatin; pipobroman; pixantrone; plicamycin; procarbazine;proteasome inhibitors (e.g.; bortezomib); raltitrexed; rebeccamycin;rubitecan; SN-38; salinosporamide A; satraplatin; streptozotocin;swainsonine; tariquidar; taxane; tegafur-uracil; temozolomide;testolactone; thioTEPA; tioguanine; topotecan; trabectedin; tretinoin;triplatin tetranitrate; tris(2-chloroethyl)amine; troxacitabine; uracilmustard; valrubicin; vinblastine; vincristine; vinorelbine; vorinostat;zosuquidar; or a combination thereof. In some embodiments, the drug is apro-apoptotic agent. In some embodiments, the drug is an anti-apoptoticagent. In some embodiments, the drug is selected from: minocycline;SB-203580 (4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)1H-imidazole); PD 169316(4-(4-Fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole); SB202190(4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole); RWJ67657(4-[4-(4-fluorophenyl)-1-(3-phenylpropyl)-5-(4-pyridinyl)-1H-imidazol-2-yl]-3-butyn-1-ol);SB 220025(542-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinlyl)imidazole);D-JNKI-1 ((D)-hJIP175-157-DPro-DPro-(D)-HIV-TAT57-48); AM-111 (Auris);SP600125 (anthra[1,9-cd]pyrazol-6(2H)-one); JNK Inhibitor I((L)-HIV-TAT48-57-PP-JBD20); JNK Inhibitor III((L)-HIV-TAT47-57-gaba-c-Junδ33-57); AS601245 (1,3-benzothiazol-2-yl(2-[[2-(3-pyridinyl) ethyl] amino]-4 pyrimidinyl) acetonitrile); JNKInhibitor VI (H2N-RPKRPTTLNLF-NH2 (SEQ ID NO: 7)); JNK Inhibitor VIII(N-(4-Amino-5-cyano-6-ethoxypyridin-2-yl)-2-(2,5-dimethoxyphenyl)acetamide);JNK Inhibitor IX(N-(3-Cyano-4,5,6,7-tetrahydro-1-benzothien-2-yl)-1-naphthamide);dicumarol (3,3′-Methylenebis(4-hydroxycoumarin)); SC-236(4-[5-[4-chlorophenyl)-3-(trifluoromethyl)-1H-pyrazol-1-yl]benzene-sulfonamide);CEP-1347 (Cephalon); CEP-11004 (Cephalon); an artificial proteincomprising at least a portion of a Bcl-2 polypeptide; a recombinant FNK;V5 (also known as Bax inhibitor peptide V5); Bax channel Blocker((±)-1-(3,6-Dibromocarbazol-9-yl)-3-piperazin-1-yl-propan-2-ol); Baxinhibiting peptide P5 (also known as Bax inhibitor peptide P5); Kp7-6;FAIM(S) (Fas apoptosis inhibitory molecule-short); FAIM(L) (Fasapoptosis inhibitory molecule-long); Fas:Fc; FAP-1; NOK2; F2051; F1926;F2928; ZB4; Fas M3 mAb; EGF; 740 Y-P; SC 3036 (KKHTDDGYMPMSPGVA (SEQ IDNO: 8)); PI 3-kinase Activator (Santa Cruz Biotechnology, Inc.); Pam3Cys((S)-(2,3-bis(palmitoyloxy)-(2RS)-propyl)-N-palmitoyl-(R)-Cys-(S)-Ser(S)-Lys4-OH,trihydrochloride); Act1 (NF-kB activator 1); an anti-IkB antibody;Acetyl-11-keto-b-Boswellic Acid; Andrographolide; Caffeic Acid PhenethylEster (CAPE); Gliotoxin; Isohelenin; NEMO-Binding Domain Binding Peptide(DRQIKIWFQNRRMKWKKTALDWSWLQTE (SEQ ID NO: 9)); NF-kB ActivationInhibitor (6-Amino-4-(4-phenoxyphenylethylamino)quinazoline); NF-kBActivation Inhibitor II(4-Methyl-N1-(3-phenylpropyl)benzene-1,2-diamine); NF-kB ActivationInhibitor III (3-Chloro-4-nitro-N-(5-nitro-2-thiazolyl)-benzamide);NF-kB Activation Inhibitor IV ((E)-2-Fluoro-4′-methoxystilbene); NF-kBActivation Inhibitor V(5-Hydroxy-(2,6-diisopropylphenyl)-1H-isoindole-1,3-dione); NF-kB SN50(AAVALLPAVLLALLAPVQRKRQKLMP (SEQ ID NO: 10)); Oridonin; Parthenolide;PPM-18 (2-Benzoylamino-1,4-naphthoquinone); Ro106-9920; Sulfasalazine;TIRAP Inhibitor Peptide (RQIKIWFNRRMKWKKLQLRDAAPGGAIVS (SEQ ID NO: 11));Withaferin A; Wogonin; BAY 11-7082((E)3-[(4-Methylphenyl)sulfonyl]-2-propenenitrile); BAY 11-7085((E)3-[(4-t-Butylphenyl)sulfonyl]-2-propenenitrile); (E)-Capsaicin;Aurothiomalate (ATM or AuTM); Evodiamine; Hypoestoxide; IKK InhibitorIII (BMS-345541); IKK Inhibitor VII; IKK Inhibitor X; IKK Inhibitor II;IKK-2 Inhibitor IV; IKK-2 Inhibitor V; IKK-2 Inhibitor VI; IKK-2Inhibitor (SC-514); IkB Kinase Inhibitor Peptide; IKK-3 Inhibitor IX;ARRY-797 (Array BioPharma); SB-220025(5-(2-Amino-4-pyrimidinyl)-4-(4-fluorophenyl)-1-(4-piperidinlyl)imidazole);SB-239063(trans-4-[4-(4-Fluorophenyl)-5-(2-methoxy-4-pyrimidinyl)-1H-imidazol-1-yl]cyclohexanol);SB-202190(4-(4-Fluorophenyl)-2-(4-hydroxyphenyl)-5-(4-pyridyl)1H-imidazole);JX-401 (-[2-Methoxy-4-(methylthio)benzoyl]-4-(phenylmethyl)piperidine);PD-169316(4-(4-Fluorophenyl)-2-(4-nitrophenyl)-5-(4-pyridyl)-1H-imidazole);SKF-86002 (6-(4-Fluorophenyl)-2,3-dihydro-5-(4-pyridinyl)imidazo[2,1-b]thiazole dihydrochloride); SB-200646(N-(1-Methyl-1H-indol-5-yl)-N′-3-pyridinylurea); CMPD-1(2′-Fluoro-N-(4-hydroxyphenyl)-[1,1′-biphenyl]-4-butanamide); EO-1428((2-Methylphenyl)-[4-[(2-amino-4-bromophl)nyeamino]-2-chlorophenyl]methanone);SB-253080(4-[5-(4-Fluorophenyl)-244-(methylsulfonyl)phenyl]-1H-imidazol-4-yl]pyridine);SD-169 (1H-Indole-5-carboxamide); SB-203580(4-(4-Fluorophenyl)-2-(4-methylsulfinyl phenyl)-5-(4-pyridyl)1H-imidazole); TZP-101 (Tranzyme Pharma); TZP-102 (Tranzyme Pharma);GHRP-6 (growth hormone-releasing peptide-6); GHRP-2 (growthhormone-releasing peptide-2); EX-1314 (Elixir Pharmaceuticals); MK-677(Merck); L-692,429 (Butanamide,3-amino-3-methyl-N-(2,3,4,5-tetrahydro-2-oxo-1-((2′-(1H-tetrazol-5-yl)(1,1′-biphenyl)-4-yl)methyl)-1H-1-benzazepin-3-yl)-,(R)-); EP1572 (Aib-DTrp-DgTrp-CHO); diltiazem; metabolites of diltiazem;BRE (Brain and Reproductive organ-Expressed protein); verapamil;nimodipine; diltiazem; omega-conotoxin; GVIA; amlodipine; felodipine;lacidipine; mibefradil; NPPB (5-Nitro-2-(3-phenylpropylamino)benzoicAcid); flunarizine; erythropoietin; piperine; hemin; brazilin; z-VAD-FMK(Benzyloxycarbonyl-Val-Ala-Asp(OMe)-fluoromethylketone); z-LEHD-FMK(benzyloxycarbonyl-Leu-Glu(OMe)-His-Asp(OMe)-fluoromethylketone) (SEQ IDNO: 12); B-D-FMK (boc-aspartyl(Ome)-fluoromethylketone); Ac-LEHD-CHO(N-acetyl-Leu-Glu-His-Asp-CHO) (SEQ ID NO: 13); Ac-IETD-CHO(N-acetyl-Ile-Glu-Thr-Asp-CHO) (SEQ ID NO: 14); z-IETD-FMK(benzyloxycarbonyl-Ile-Glu(OMe)-Thr-Asp(OMe)-fluoromethylketone) (SEQ IDNO: 15); FAM-LEHD-FMK (benzyloxycarbonyl Leu-Glu-His-Asp-fluoromethylketone) (SEQ ID NO: 16); FAM-LETD-FMK (benzyloxycarbonylLeu-Glu-Thr-Asp-fluoromethyl ketone) (SEQ ID NO: 17); Q-VD-OPH(Quinoline-Val-Asp-CH2-O-Ph); XIAP; cIAP-1; cIAP-2; ML-IAP; ILP-2; NAIP;Survivin; Bruce; IAPL-3; fortilin; leupeptine; PD-150606(3-(4-Iodophenyl)-2-mercapto-(Z)-2-propenoic acid); MDL-28170(Z-Val-Phe-CHO); calpeptin; acetyl-calpastatin; MG 132(N-[(phenylmethoxy)carbonyl]-L-leucyl-N-[(1S)-1-formyl-3-methylbutyl]-L-leucinamide);MYODUR; BN 82270 (Ipsen); BN 2204 (Ipsen); AHLi-11 (QuarkPharmaceuticals), an mdm2 protein, pifithrin-α(1-(4-Methylphenyl)-2-(4,5,6,7-tetrahydro-2-imino-3(2H)-benzothiazolyl)ethanone);trans-stilbene; cis-stilbene; resveratrol; piceatannol; rhapontin;deoxyrhapontin; butein; chalcon; isoliquirtigen; butein;4,2′,4′-trihydroxychalcone; 3,4,2′,4′,6′-pentahydroxychalcone; flavone;morin; fisetin; luteolin; quercetin; kaempferol; apigenin; gossypetin;myricetin; 6-hydroxyapigenin; 5-hydroxyflavone;5,7,3′,4′,5′-pentahydroxyflavone; 3,7,3′,4′,5′-pentahydroxyflavone;3,6,3′,4′-tetrahydroxyflavone; 7,3′,4′,5′-tetrahydroxyflavone;3,6,2′,4′-tetrahydroxyflavone; 7,4′-dihydroxyflavone;7,8,3′,4′-tetrahydroxyflavone; 3,6,2′,3′-tetrahydroxyflavone;4′-hydroxyflavone; 5-hydroxyflavone; 5,4′-dihydroxyflavone;5,7-dihydroxyflavone; daidzein; genistein; naringenin; flavanone;3,5,7,3′,4′-pentahydroxyflavanone; pelargonidin chloride; cyanidinchloride; delphinidin chloride; (−)-epicatechin (Hydroxy Sites:3,5,7,3′,4′); (−)-catechin (Hydroxy Sites: 3,5,7,3′,4′);(−)-gallocatechin (Hydroxy Sites: 3,5,7,3′,4′,5′) (+)-catechin (HydroxySites: 3,5,7,3′,4′); (+)-epicatechin (Hydroxy Sites: 3,5,7,3′,4′);Hinokitiol (b-Thujaplicin;2-hydroxy-4-isopropyl-2,4,6-cycloheptatrien-1-one); L-(+)-Ergothioneine((S)-a-Carboxy-2,3-dihydro-N,N,N-trimethyl-2-thioxo-1H-imidazole4-ethanaminiuminner salt); Caffeic Acid Phenyl Ester; MCI-186(3-Methyl-1-phenyl-2-pyrazolin-5-one); HBED(N,N′-Di-(2-hydroxybenzyl)ethylenediamine-N,N′-diacetic acid.H2O);Ambroxol (trans-4-(2-Amino-3,5-dibromobenzylamino)cyclohexane-HCl; andU-83836E((−)-2-((4-(2,6-di-1-Pyrrolidinyl-4-pyrimidinyl)-1-piperzainyl)methyl)-3,4-dihydro-2,5,7,8-tetramethyl-2H-1-benzopyran-6-ol.2HCl);β-1′-5-methyl-nicotinamide-2′-deoxyribose;β-D-1′-5-methyl-nicotinamide-2′-deoxyribofuranoside;β-1′-4,5-dimethyl-nicotinamide-2′-de-oxyribose;β-D-1′-4,5-dimethyl-nicotinamide-2′-deoxyribofuranoside; 1-Naphthyl PP1(1-(1,1-Dimethylethyl)-3-(1-naphthalenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine); Lavendustin A(5-[[(2,5-Dihydroxyphenyl)methyl][(2-hydroxyphenyl)methyl]amino]-2-hydroxybenzoicacid); MNS (3,4-Methylenedioxy-b-nitrostyrene); PP1(1-(1,1-Dimethylethyl)-1-(4-methylphenyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine); PP2 (3-(4-chlorophenyl)1-(1,1-dimethylethyl)-1H-pyrazolo[3,4-d]pyrimidin-4-amine); KX-004(Kinex); KX-005 (Kinex); KX-136 (Kinex); KX-174 (Kinex); KX-141 (Kinex);KX2-328 (Kinex); KX-306 (Kinex); KX-329 (Kinex); KX2-391 (Kinex);KX2-377 (Kinex); ZD4190 (Astra Zeneca;N-(4-bromo-2-fluorophenyl)-6-methoxy-7-(2-(1H-1,2,3-triazol-1-yl)ethoxy)quinazolin-4-amine);AP22408 (Ariad Pharmaceuticals); AP23236 (Ariad Pharmaceuticals);AP23451 (Ariad Pharmaceuticals); AP23464 (Ariad Pharmaceuticals);AZD0530 (Astra Zeneca); AZM475271 (M475271; Astra Zeneca); Dasatinib(N-(2-chloro-6-methylphneyl)-2-(6-(4-(2-hydroxyethyl)-piperazin-1-yl)-2-methylpyrimidin-4-ylamino)thiazole-5-carboxamide); GN963 (trans-4-(6,7-dimethoxyquinoxalin-2ylamino)cyclohexanol sulfate); Bosutinib(4-((2,4-dichloro-5-methoxyphenyl)amino)-6-methoxy-7-(3-(4-methyl-1-piperazinyl)propoxy)-3-quinolinecarbonitrile);or combinations thereof.

Pharmaceutical Compositions

Disclosed herein, in certain embodiments, are pharmaceuticalcompositions comprising a selective transport molecule disclosed herein.Pharmaceutical compositions herein are formulated using one or morephysiologically acceptable carriers including excipients and auxiliarieswhich facilitate processing of the active agents into preparations whichare used pharmaceutically. Proper formulation is dependent upon theroute of administration chosen. A summary of pharmaceutical compositionsis found, for example, in Remington: The Science and Practice ofPharmacy, Nineteenth Ed (Easton, Pa.: Mack Publishing Company, 1995);Hoover, John E., Remington's Pharmaceutical Sciences, Mack PublishingCo., Easton, Pa. 1975; Liberman, H. A. and Lachman, L., Eds.,Pharmaceutical Dosage Forms, Marcel Decker, New York, N.Y., 1980; andPharmaceutical Dosage Forms and Drug Delivery Systems, Seventh Ed.(Lippincott Williams & Wilkins, 1999).

In certain embodiments, a pharmaceutical composition disclosed hereinfurther comprises a pharmaceutically acceptable diluent(s),excipient(s), or carrier(s). In some embodiments, the pharmaceuticalcompositions includes other medicinal or pharmaceutical agents,carriers, adjuvants, such as preserving, stabilizing, wetting oremulsifying agents, solution promoters, salts for regulating the osmoticpressure, and/or buffers. In addition, the pharmaceutical compositionsalso contain other therapeutically valuable substances.

In certain embodiments, a pharmaceutical composition disclosed herein isadministered to a subject by any suitable administration route,including but not limited to, parenteral (intravenous, subcutaneous,intraperitoneal, intramuscular, intravascular, intrathecal,intravitreal, infusion, or local) administration.

Formulations suitable for intramuscular, subcutaneous, or intravenousinjection include physiologically acceptable sterile aqueous ornon-aqueous solutions, dispersions, suspensions or emulsions, andsterile powders for reconstitution into sterile injectable solutions ordispersions. Examples of suitable aqueous and non-aqueous carriers,diluents, solvents, or vehicles including water, ethanol, polyols(propyleneglycol, polyethylene-glycol, glycerol, cremophor and thelike), suitable mixtures thereof, vegetable oils (such as olive oil) andinjectable organic esters such as ethyl oleate. Proper fluidity ismaintained, for example, by the use of a coating such as lecithin, bythe maintenance of the required particle size in the case ofdispersions, and by the use of surfactants. Formulations suitable forsubcutaneous injection also contain optional additives such aspreserving, wetting, emulsifying, and dispensing agents.

For intravenous injections, an active agent is optionally formulated inaqueous solutions, preferably in physiologically compatible buffers suchas Hank's solution, Ringer's solution, or physiological saline buffer.

Parenteral injections optionally involve bolus injection or continuousinfusion. Formulations for injection are optionally presented in unitdosage form, e.g., in ampoules or in multi dose containers, with anadded preservative. In some embodiments, the pharmaceutical compositiondescribed herein are in a form suitable for parenteral injection as asterile suspensions, solutions or emulsions in oily or aqueous vehicles,and contain formulatory agents such as suspending, stabilizing and/ordispersing agents. Pharmaceutical formulations for parenteraladministration include aqueous solutions of an active agent in watersoluble form. Additionally, suspensions are optionally prepared asappropriate oily injection suspensions.

In some embodiments, the pharmaceutical composition described herein isin unit dosage forms suitable for single administration of precisedosages. In unit dosage form, the formulation is divided into unit dosescontaining appropriate quantities of an active agent disclosed herein.In some embodiments, the unit dosage is in the form of a packagecontaining discrete quantities of the formulation. Non-limiting examplesare packaged tablets or capsules, and powders in vials or ampoules. Insome embodiments, aqueous suspension compositions are packaged insingle-dose non-reclosable containers. Alternatively, multiple-dosereclosable containers are used, in which case it is typical to include apreservative in the composition. By way of example only, formulationsfor parenteral injection are presented in unit dosage form, whichinclude, but are not limited to ampoules, or in multi dose containers,with an added preservative.

EXAMPLES Example 1: Peptide Synthesis

A number of peptides whose cell uptake could be modulated weresynthesized. The following symbols, where used, are used with theindicated meanings: Fl=fluorescein aca=ahx=X=aminohexanoyl linker(—HN—(CH2)5-CO-)aminohexanoyl, C=L-cysteine, E=L-glutamate,R=L-arginine, D=L-aspartate, K=L-lysine, A=L-alanine, r=D-arginine,c=D-cysteine, e=D-glutamate, P=L-proline, L=L-leucine, G=glycine,V=valine, I=isoleucine, M=methionine, F=phenylalanine, Y=tyrosine,W=tryptophan, H=histidine, Q=glutamine, N=asparagine, S=serine,T=threonine, o is 5-amino-3-oxapentanoyl linker, and C(me) isS-methylcysteine.

In sequences discussed below, lower case letters indicate the D isomerof the amino acid.

Peptides were synthesized on a peptide synthesizer (Pioneer PeptideSynthesis System by Applied Biosystems) using solid phase synthesismethod and commercial available Fmoc amino acids, resins, and the otherreagents. The peptides were cleaved withTFA/thioanisole/triisopropylsilane orTFA/thioanisole/triisopropylsilane/ethanedithiol.

Peptides were labeled with 5-(and-6)carboxyfluorescein succinimidylester on the amino group on the peptide or with5-iodoacetamidofluorescein on the thiol group on the peptide.

The crude peptide was purified on HPLC and lyophilized overnight.

Each peptide composition was confirmed by mass spectrometry.

Synthesis of a Selective Transport Molecule (Hereinafter, Peptide 1)

Suc-e₈-(Aop)-PLGC(me)AG-r₉-c-NH₂ was synthesized via standard Fmoc solidphase peptide synthesis. The N-terminal succinyl group was added to thepeptide by reaction with succinic anhydride while still on resin. Thepeptide was cleaved from the resin in a standard cocktail(trifluoroacetic acid with 2% each of thioanusole, triisopropylsilaneand ethandithiol) overnight at room temperature. Most of thetrifluoroacetic acid was remove by rotary evaporator, 50% hexanes indiethyl ether was added and the peptide was collected by centrifugation.The collected solid was washed with 50% hexanes in ether three times andvacuum dried overnight. The peptide was purified on HPLC using 15%-30%acetonitrile in water and 0.05% TFA, giving a 30% yield from the crudepeptide. The correct purified product was confirmed by electrospray massspectroscopy: calculated 3271.5 Da, found 3271.8 Da.

Synthesis of Peptide-Labeled Dendrimer 2

25 mg of peptide 1 was dissolved in 2 mL DMSO under N₂ and was reactedwith 2.3 mg 2-nitro-4-sulfophenyl 6-maleimidohexanoate sodium salt and20 μL N-methylmorpholine. After stirring at room temperature for threehours, LC-MS analysis of the reaction mixture indicated over 90%completion. The reaction mixture was cooled to 0°, 150 mg PAMAMdendrimer and 2 mL 1 M Hepes buffer (pH 7.8) were added and stirred at5° for 2 days (hereinafter, reaction mixture 2).

Synthesis of Cy5- and Peptide-Labeled Dendrimer 3.

1.2 mg Cy5 mono(N-hydroxysuccinimide) was added to reaction mixture 2and stirred at 5° overnight (hereinafter, reaction mixture 3).

Synthesis of Capped Cy5- and Peptide-Labeled Dendrimer 4.

166 mg of MeO(CH₂CH₂O)₃CH₂CH₂CO—(N-hydroxysuccinimide)ester was added toreaction mixture 3 at 5° and stirred at that temperature for 3 days. Thecrude product was diluted with 10 mL of water, and low molecular weightcontaminants were removed by filtration 8 times through a membrane witha 10 kDa cutoff. HPLC using a size-exclusion column indicated 99%purity, 72% yield. An average of 3 fluorophores per dendrimer wasdetermined by dissolving a known weight of purified final product inwater and measuring Cy5 absorbance at 650 nm, assuming an extinctioncoefficient of 250,000M⁻¹ cm⁻¹. Static multiangle light-scattering at785 nm indicated an apparent molecule weight of 72.9 kDa. Dynamic lightscattering at 785 nm indicated a hydrodynamic radius of 4.6 nm.

Synthesis of DOTA-, Cy5- and Peptide-Labeled Dendrimer 5.

30 equivalents of DOTA mono-N-hydroxysuccinimde ester in HEPES bufferwere reacted with reaction mixture 3 and stirred at 5° overnight(hereinafter, reaction mixture 5).

Synthesis of Capped DOTA-, Cy5- and Peptide-Labeled Dendrimer 6.

Reaction mixture 5 was reacted with 950 equivalents mPEG4 NHS andstirred at 5° for three days. The crude product was purified asdescribed for capped Cy5- and peptide-labeled dendrimer 4, thenlyophilized. The yield was 78%.

Gd Loading of Capped DOTA-, Cy5- and Peptide-Labeled Dendrimer 6

25 mg of capped DOTA-, Cy5- and peptide-labeled dendrimer 6 wasdissolved in 1 mL 0.5M ammonium acetate and 1 mL water. The reactionmixture was mixed with 100 μL 0.5 GdCl₃ and stirred at room temperaturefor 3 days shielded from light. Small molecules were eliminated by 5aqueous washes. Excess water was removed by centrifugation through amembrane filter with a 10 kDa cutoff. Finally, the Gd loaded product 7was lyophilized overnight to give a blue fluffy solid. The pure productwas weighed and redissolved in water to give a 200 uM solution. Ameasured small aliquot was mixed with 0.5 mL concentrated nitric acidfor 2 hours. Gd quantitation was determined by inductively coupledplasma mass spectroscopy, which indicated an average of 15 Gd perdendrimer. The number of Cy5 labels per dendrimer was confirmed to be 3based on 650 nm absorbance.

Example 2: In Vivo Fluorescence Imaging of Atherosclerotic Plaques withSelective Transport Molecules

Peptide Synthesis

Briefly, peptides were synthesized on an automatic peptide synthesizerfollowing standard procedures for fluorenylmethoxycarbonyl solid-phasesynthesis. Peptides were N-terminally capped with a succinate andC-termini were amidated. After cleavage off the resin, the C-terminiwere labeled through the cysteine with Cy5or rhodamine monomaleimide.Peptides were purified using HPLC. Specific peptide compositions were:DPRSFL (SEQ ID NO: 1)-based transport molecule=Suc-e9-O-DPRSFL-r9-c(Cy5or rhodamine)-CONH2), DPRSFL (SEQ ID NO: 1)-based transportmolecule=(Suc-e9-ahx-dprsfl-r9-c(Cy5)-CONH2), and mPEG-selectivetransport molecule=(Suc-e9-(mPEG)-r9-c(Cy5)-CONH2) whereahx=aminohexanoic acid, 0=5-amino-3-oxapentanoyl and mPEG=(PEG₂)₂. Lowercase letters represent D-amino acids.

Determination of k_(cat)/k_(m)

Reactions containing 50 nM thrombin, and seven peptide concentrationsranging from 1 to 30 μM DPRSFL-based transport molecule peptide wereincubated for 5, 15, 30 and 60 minutes in 100 μL reaction volumes beforeundergoing tricine gel electrophoresis. Reactions were stopped byaddition of SDS containing sample buffer at the appropriate time point.Percent cleavage was assessed as before, and was multiplied by thestarting concentration for each vial to obtain the total productconcentration. The velocity of each reaction was obtained bydetermination of the slope of the linear portion of the curve on ascatterplot comparing product vs. time. The k_(cat) and k_(m) wereobtained by determining the y-intercept and slope on a Lineweaver-Burkeplot.

Serum Cleavage Experiments

Whole blood was collected either via cardiac puncture or from theabdominal aorta, in either heparinized tubes (plasma) or eppendorf tubes(serum). Both tubes were centrifuged at low speeds to pellet out the redblood cells, and the supernatant was removed and frozen for future use.Cleavage assays were done using a final concentration of between 2 and 5μM peptide. Argatroban (Enzo Life Sciences Plymouth Meeting, Pa.) andlepirudin (UCSD Hillcrest Hospital pharmacy and were used at a finalconcentration of 4 mg/mL and 0.5 mg/mL respectively. After 20 minutes,reactions were stopped by addition of SDS containing tricine samplebuffer and heating to 85° C. Cleavage was assayed by tricine gelelectrophoresis at 100V (Invitrogen, Carlsbad, Calif.), and bands werequantified by integrated density using Image J. Percent cleavage andtotal observed cleavage were calculated as before.

Methods for Enzyme Panel

Enzymes were purchased from commercial sources: recombinant plasmin,Factor VIIa, trypsin, chymotrypsin, thrombin, Factor Xla, Factor XIIa,cathepsin G, neutrophil elastase, and activated protein C (APC) werepurchased from EMD, La Jolla Calif., Factor Xa from NEB, Ipswitch,Mass., pancreatic elastase from Sigma Aldrich. 5 uM substrate wasincubated with 50 nM and rate of cleavage was determined by theemergence of a lower molecular weight band on commercially preparedtricine gel electrophoresis (Invitrogen). Band intensity was quantifiedusing Image J, with “background” defined as the average of a larger areain the middle of the gel, away from any fluorescence bands. This“background” is due to CCD noise. Percent cleavage was defined as(intensity of the lower molecular weight band minus“background”)/(intensity of lower plus higher molecular weight bandminus 2 times background). To account for minor impurities in thesample, the percent cleavage for uncleaved substrate has been subtractedoff (FIG. S1)

Ex Vivo Clot Aging Experiments

Blood was obtained from mice (n=3) or rats (n=1) via cardiac punctureand allowed to clot in an Eppendorf tube. Small clots (−30-60 mg) werecut off for processing at each time point. Clots were stored in ahumidified chamber prior to use. At the indicated time point, clots wereincubated for 10 minutes in either 2.5 and 5 μM DPRSFL (SEQ ID NO:1)-based transport molecule with or without addition of argatroban orlepirudin (4 mg/mL and 0.5 mg/mLfinal concentrations respectively) ormPEG-selective transport molecule. At least two clots were assayed foreach time point for each animal. After staining, clots were washed threetimes in 2 mL PBS with shaking. Clots were then imaged using afluorescence mouse imager (CRI, Maestro, ex 640/48, em 700 nm). Datawere quantified by taking the average intensity of the entire clot inImage J. Each condition was compared to the result for DPRSFL-basedtransport molecule alone, and significance was assessed using anunpaired, two-tailed Students t-test.

Animals and Preparation of In Vivo Clots

Wildtype FVB mice (n=12) were obtained from Charles River (Wilmington,Mass.). Tails were amputated 2.5 mm from the tip. Following tailamputation, 10 nmol of either DPRSFL-based transport molecule ormPEG-selective transport molecule was injected intravenously. Mice wereanesthesitized with ketamine/midazolam (80 mg/kg, 5 mg/kg) and tailswere imaged using a Zeiss Lumar dissecting microscope (ex 620/60, em700/75, 0.8× objective, Zeiss, Peabody, Mass.). Significance wasassessed using an unpaired two-tailed Students t-test. All animalprocedures were approved by UCSD's institutional animal care and usecommittee.

Atherosclerosis Models

LDLR^(−/−) or ApoE^(−/−) mice were fed a high cholesterol diet (0.5-2%)for 1-2 years prior to the experiment, generating animals with moderateto severe atherosclerotic lesions[42]. Animals were used once they wereabout to be sacrificed for health reasons ranging from skin ulcerationsto the sudden overnight death of cage-mates. For a more completebreakdown of the study population including ages, duration of diet,gender and genotype, see Table S1 and S2 Inhibitors used wererecombinant hirudin at 2000 U/mouse (EMD Biosciences), and a cocktail ofSB3CT (500 μg/animal) and GM6001 (2 mg/animal).

Imaging of Aortas

Animals were injected with either mPEG-selective transport molecule orDPRSFL-based transport molecule with or without pre-injection 250 μgrecombinant hirudin (EMD Biosciences). Probe was allowed to circulatefor six hours. To obtain aorta for imaging of whole vessel fluorescence,mice were transcardially perfused with saline followed by bufferedformaldehyde. Whole aorta were dissected from the level of the aorticarch to the abdominal aorta and then bisected for pinning onto wax forfluorescent imaging of bound selective transport molecules using aMaestro Imager™ ((700 nm) using a 640/48 excitation filter) (Cri, WoburnMass.). Percent plaque and plaque intensity were calculated using Amirasoftware (Visage Imaging, La Jolla, Calif.), FIG. S1. To account forday-to-day variation in light levels, for each experiment plaqueintensity is reported as raw plaque intensity/phantom intensity taken onthe same day (Labsphere certified reflectance standard, North Sutton,N.H.).

AHA Plaque Stage Determination

For histopathology preparation, aortic arch samples with plaquediscernable with white light illumination were collected andcryoprotected in 30% (w/v) buffered sucrose. Samples were frozen inTissue-Tek embedding medium (Torrance Calif.). Each block was sectionedat 10 μm thickness and sections were thaw mounted onto glass slides;adjacent slides were stained with hematoxylin/eosin (H/E) or with theGomori Trichrome method. Slides were imaged dry on a Zeiss Lumardissecting microscope (ex 620/60, em 700/75, 1.5x objective).Histological interpretation and categorization of arterial sectionsincluding the vessel wall was performed by a blinded, board certifiedpathologist (SB). Significance between results from AHA Class 5 vs. AHAClass 6 was determined by two tailed, unpaired Students t-test.

Whole Animal Imaging

Animals were briefly anesthetized with ketamine/midazolam (80 mg/kg, 5mg/kg) and co-injected with the indicated selective transportmolecule(s). 18-24 hours later, animals were anesthetized by injectionof ketamine (80 mg/kg) and midazolam (5 mg/kg) or euthanized by anoverdose of the same two drugs. The carotid artery was exposedpre-mortem and was imaged using a custom surgical apparatus in our lab.Other structures including the carotid bifurcation and the aortic archwere exposed postmortem and were imaged using a Zeiss Lumar dissectingmicroscope (ex 560/25, em 607/36 for rhodamine, ex 620/60, em 700/75 forCy5, 0.8x objective, Zeiss, Peabody, Mass.). After imaging, tissue wasfrozen on dry ice for histology.

Fresh Frozen Tissue Histology

Frozen tissue was cut to 10 μm and imaged dry using a Zeiss Lumardissecting microscope ex 560/25, em 607/36 for rhodamine, ex 620/60, em700/75 for Cy5, 1.5x objective). Overlays were done using Metamorphsoftware (Molecular Devices, Sunnyvale, Calif.).

Immunocytochemistry

Mice were euthanized with an overdose of ketamine-midazolam.

Immunochemistry was performed on fresh aorta specimen dissected fromeuthanized mice and immediately snap frozen with dry ice in Tissue-tekfilled molds. Cryostat sections (10 μm thick) were incubated withanti-F4/80 (clone BM8) (14-4801-81 e-Bioscience, San Diego) to visualizemacrophages. For identification of macrophages as well as other myeloidcells, sections were incubated with antibodies to CD68 (ab 53444-100,Abcam, Cambridge, Mass.). After a 48 hour incubation the bound primaryantibody was visualized with biotinylated secondary antibody (AP183B,Millipore, Temecula Ca) followed by treatment with avidin-biotinylatedperoxidase (RTU Vectastain, Vector labs Burlingame Calif.) anddiaminobenizdine (Vector labs, Burlingame Calif.). Alternate sectionsthat were not immunoreacted were imaged with epifluorescence microscopyto visualize the distribution of fluorescent selective transportmolecule.

Human Specimen

Postoperative human endarterectomy specimens were obtained at UCSD fromDrs. Nikhil Kansal, Erik Owens and Katherine Brown (UCSD IRB#080965).Specimens were transported from the operating room to our lab inapproximately 5 minutes, where they were embedded in 0.5% agarose geland cut into 2 mm sections. Sections were incubated in a 96-well platefor 1 hour in 10 mM of either DPRSFL (SEQ ID NO: 1)-based transportmolecule, PLGLAG (SEQ ID NO: 4)-selective transport molecule ormPEG-selective transport molecule as described above and washed sixtimes for 15 minutes in PBS prior to embedding in Cryotec and freezingat −80 C. 8 nm cryostat sections were cut and mounted onto glass slides.Imaging was done using the Zeiss Lumar microscope (ex 620/60, em 700/75,0.8x objective, 8× magnification). Data was collected using MetaMorphsoftware version 6.1 (Silicon Valley, Calif.). Quantification offluorescence intensity was performed using ImageJ software (NIH,Bethesda, Md.). Average background fluorescence and fluorescenceintensity were measured and background subtracted (FIG. S6). Since thespecimens were collected over 6-8 months and the same atheromas wereused to test experimental and control selective transport molecules,both unpaired and matched-paired t-tests were used to compare relativefluorescence intensity, with significance defined as p<0.05.

DPRSFL (SEQ ID NO: 1)-Based Transport Molecule is a Novel SelectiveTransport Molecule that is Cleavable by Thrombin and Serum Derived fromWhole Blood.

We first used purified thrombin to determine whether two candidatethrombin cleavage sites found in the literature would be selective forthrombin once bent into the typical selective transport molecule hairpinconfiguration (14). The first cleavage site tested, Norleucine-TPR, camefrom a screen for fluorogenic substrates for thrombin using a positionalscanning library (32) and was barely cleavable by purified thrombinafter insertion into the selective transport molecule hairpin. Thesecond, DPRSFL (SEQ ID NO: 1), is comprised of amino acids 39-44 of thePAR-1 thrombin receptor (FIG. 1A,28). Since activation of this receptorby thrombin through cleavage of the DPRSFL (SEQ ID NO: 1) sequence isinvolved in a wide range of pathological processes, we hypothesized thatthis new selective transport molecule should be reasonably selective forthrombin activity in vivo. As expected, DPRSFL (SEQ ID NO: 1)-basedtransport molecule was efficiently cleaved by purified thrombin in vitrowith a k_(cat/km) of 2.1×10⁴. This is slower than thrombin cleavage ofthe native PAR-1 receptor (k_(cat)/k_(m) of 7.6×10⁶(26)), likely due tobending of the substrate sequence secondary to the hairpin shapedselective transport molecule (14) and removal of the hirudin bindingdomain (27). Incubation in active serum also resulted in rapid cleavageof the DPRSFL (SEQ ID NO: 1)-based transport molecule. Serum cleavagewas completely inhibitable by thrombin inhibitors such as lephirudin andargatroban, suggesting that thrombin is responsible for cleavage even inthis complex mixture of coagulation enzymes. No cleavage was seen uponincubation of the DPRSFL (SEQ ID NO: 1)-based transport molecule inplasma, or anticoagulated blood that should not contain active thrombin(FIG. 1B, FIG. S1)

DPRSFL (SEQ ID NO: 1)-Based Transport Molecule is Cleaved by SeveralCommercially Available Enzymes Known to Cleave the PAR-1 Receptor.

Several other enzymes are known to cleave the PAR-1 receptor in vitro,though less efficiently than thrombin. To determine the specificity ofthe DPRSFL (SEQ ID NO: 1) cleavage site for thrombin, a panel ofcommercially available enzymes was tested for cleavage of DPRSFL (SEQ IDNO: 1)-based transport molecule. As expected, several of these enzymesknown to cleave the native PAR-1 receptor at the DPRSFL (SEQ ID NO: 1)cleavage site were able to cleave the DPRSFL (SEQ ID NO: 1)-basedtransport molecule, including thrombin, plasmin, Factor Xa, trypsin andchymotrypsin. (FIG. 1C). In separate tests, coagulation factors VIIa,IXa, XIa and XIIa, elastase, MMP's 2, 7 and 9, cathepsin, renin, uPA andtPA did not cleave the DPRSFL (SEQ ID NO: 1)-based transport moleculeefficiently. Taken together with the inhibitor data shown previously,these data support the hypothesis that thrombin is most likelyresponsible for cleavage of DPRSFL (SEQ ID NO: 1)-based transportmolecule in active serum and confirm that direct thrombin inhibitors area useful method for verifying that selective transport molecule cleavageand uptake is due to thrombin in new model systems.

DPRSFL (SEQ ID NO: 1)-Based Transport Molecule is Cleaved and Taken Upinto Blood Clots Using Either In Vitro or In Vivo Clot Models.

To determine whether DPRSFL (SEQ ID NO: 1)-based transport moleculecleavage caused uptake of the cleaved fragment into blood clots, freshclots were created postmortem by allowing blood to clot onto glass.Similarly sized clots were incubated in DPRSFL (SEQ ID NO: 1)-basedtransport molecule with and without direct thrombin inhibitors andlabeling was compared to that of a control selective transport moleculecontaining an inert (PEG₂)₂ linker replacing the DPRSFL (SEQ ID NO: 1)cleavage site (mPEG-selective transport molecule, 15). Since selectivetransport molecule's have no quenching mechanism, it was necessary towash clots extensively after incubation in probe to disperse uncleavedselective transport molecule. The DRPSFL (SEQ ID NO: 1)-selectivetransport molecule showed between a two and six-fold increase in signalwhen compared to uptake of the control selective transport molecule.Uptake of DPRSFL (SEQ ID NO: 1)-based transport molecule was completelyinhibitable by addition of either lephirudin or argatroban prior to clotincubation with probe (FIG. S2). In a separate set of experiments, an invivo tail amputation model [33] was used to test whether the DPRSFL (SEQID NO: 1)-based transport molecule highlighted clots formed in vivo. Weinjected 10 nmols of either DPRSFL (SEQ ID NO: 1)-based transportmolecule or mPEG-selective transport molecule twenty minutes or sixhours following tail amputation. Uptake of DPRSFL (SEQ ID NO: 1)-basedtransport molecule was 1.9 to 2.4-fold greater than mPEG-selectivetransport molecule (FIG. S3), with no significant difference in uptakebetween time points.[[.]]

DPRSFL (SEQ ID NO: 1)-Based Transport Molecule and PLGLAG (SEQ ID NO:4)-Selective Transport Molecule Uptake was Enzyme Dependent andCorrelated with Increased Plaque Burden in Atherosclerotic Mice.

Atherosclerosis was studied in aged transgenic mice deficient in eitherApoE (ApoE^(−/−)) or LDLR (LDLR^(−/−)) that had been maintained on aprolonged high-fat diet. Many of the animals with higher plaque burdenwere clinically ill at time of use, and their disease had progressed tosuch a point that they would have been sacrificed for health reasons hadthey not been included in this study. Animals were injected with DPRSFL(SEQ ID NO: 1)-based transport molecule alone (FIG. 2A), DPRSFL (SEQ IDNO: 1)-based transport molecule with pre-injection of hirudin (FIG. 2B),MMP cleavable PLGLAG (SEQ ID NO: 4)-selective transport molecule (FIG.2C) or with negative control peptide mPEG-selective transport molecule(FIG. 2D). Representative plaques with Gamori trichrome stains onadjacent sections are shown for DPRSFL (SEQ ID NO: 1)-based transportmolecule and PLGLAG (SEQ ID NO: 4)-selective transport molecule in FIG.2E-H. Gross fluorescence uptake was quantitated for whole dissectedaortas six hours after injection with selective transport molecule. BothDPRSFL (SEQ ID NO: 1)-based transport molecule and PLGLAG (SEQ ID NO:4)-selective transport molecule showed a quantitative increase thatcorrelated moderately well with total plaque burden (correlationcoefficient 0.8 and 0.85 respectively, FIG. 2E, FIG. S4). For thepurpose of analysis, aortas were stratified into three groups based ontheir overall plaque burden, low (0-30%), medium (30-55%) and high(>55%). We found that while uptake of DPRSFL (SEQ ID NO: 1)-basedtransport molecule and PLGLAG (SEQ ID NO: 4)-selective transportmolecule were similar when plaque burden was low (5.7±2.7 vs. 5.9±2.9,p=0.98), DPRSFL (SEQ ID NO: 1)-based transport molecule was taken upmore in animals with high plaque burden (19.9±8.3 vs. 14.7±1.5, p=0.24)FIG. 2A, 2B, 2E, FIG. 51). Hirudin was effective at inhibiting uptake athigh plaque burden, with 85% inhibition when compared to the mPEGcontrol selective transport molecule as baseline. By comparison, MMPinhibitors such as SB3CT and GM6001 reduced uptake of PLGLAG (SEQ ID NO:4)-selective transport molecule by only 30% in animals with high plaqueburden, consistent with previous results using the PLGLAG (SEQ ID NO:4)-selective transport molecule's in tumors (15). There was very littleuptake in the arterial wall of a single wildtype mouse (0.25.), comparedto uptake in the grossly plaque free aortic wall in its LDLR and ApoEdeficient counterparts (2.2±1.8 a.u.). In contrast, uptake of PLGLAG(SEQ ID NO: 4)-selective transport molecule into the arterial wall of asingle wildtype mouse was similar to that of grossly plaque free aorticwall in its LDLR and ApoE deficient counterparts (2.6 vs. 3.0±1.3 a.u.).

Thrombin Specific Uptake is Elevated in High Risk Lesions, Whereas MMPSpecific Uptake is not.

To determine whether high risk lesions correlated with increasedthrombin and MMP activity, a representative section of visible plaquefrom the aortic arch of each mouse was sectioned and stained with H/Eand Gomori trichrome. All stained plaques were categorized in a blindedfashion by a board-certified pathologist into AHA Class 1-6 (2). Thevast majority of the plaques were either AHA Class 5 (fibrous plaque,FIGS. 3A and B, FIG. 3E) or AHA Class 6 (complicated lesion/rupture,FIGS. 3C and D, FIG. 3F). These AHA category assignments were then usedto stratify the gross fluorescence intensity data obtained from wholeaortas into those containing higher risk plaques and those containinglower risk plaques. We found that aortas with the highest gross DPRSFL(SEQ ID NO: 1)-based transport molecule uptake were those for which therepresentative plaque was categorized as a high risk AHA Class 6(24.4±3.6, n=4). Three out of four of these plaques were from animalsthat were also in the high plaque group when animals were stratified byplaque burden. In contrast, lower risk AHA Class 5 plaques demonstratedsignificantly less DPRSFL (SEQ ID NO: 1)-based transport molecule uptake(7.8±3.0, n=12, p=0.0007, FIG. 3G). In contrast, gross PLGLAG (SEQ IDNO: 4)-selective transport molecule uptake was statistically similarbetween cases with AHA Class 5 (11.4±3.4, n=5) or AHA Class 6 (11.5±4.2,n=3) p=0.97, FIG. 3H).

Localization of Thrombin and MMP Activity is Distinct and VariesThroughout the Course of Plaque Development.

To examine differences between localization of thrombin and MMP activitythroughout the course of plaque development, two LDLR^(−/−) mice wereco-injected with tetramethylrhodamine isothiocyanate (TRITC) labeledDPRSFL (SEQ ID NO: 1)-based transport molecule and Cy5-PLGLAG (SEQ IDNO: 18)-selective transport molecule and sacrificed without perfusion14-18 hours after injection (FIGS. 4A and B). Following dual wavelengthgross imaging, aortas were cryosectioned and examined using dualwavelength fluorescence microscopy. Both mice had extensive, nearocclusive, calcified plaque in the aortic arch with histologicalevidence of rupture in addition to less severe atherosclerotic diseasein the descending thoracic aorta. Earlier plaques from the descendingthoracic aorta frequently took up DPRSFL (SEQ ID NO: 1)-based transportmolecule, but not PLGLAG (SEQ ID NO: 4)-selective transport molecule(FIG. 4C-E). A common finding in more advanced, fibrosing plaques wasdiffuse uptake of the DPRSFL (SEQ ID NO: 1)-based transport moleculethroughout blue fibrotic regions seen on trichrome staining of adjacentsections (FIG. 1E) but not in lipid laden macrophages at the surface ofplaques (FIG. S5). Finally, both PLGLAG (SEQ ID NO: 4)-selectivetransport molecule and DPRSFL (SEQ ID NO: 1)-based transport moleculewere frequently taken up into discrete puncta with occasionalco-localization (FIG. 4F-K). These puncta did not colocalize with F480expressing macrophages, although there was some colocalization with theless specific CD68 antigen (FIG. S5). PLGLAG (SEQ ID NO: 4) and DPRSFL(SEQ ID NO: 1)-based transport molecule's detect enzymatic activity inhuman atheromas.

Seven atheroma specimen were obtained following carotid endarterectomy.Each specimen was cut into 1-2 mm slices that were then incubated ineither PLGLAG (SEQ ID NO: 4)-selective transport molecule, DPRSFL (SEQID NO: 1)-based transport molecule or mPEG-selective transport molecule.The slices were then cryosectioned to look at selective transportmolecule uptake in the interior of the plaque. Qualitatively, we foundthat specimens incubated in DPRSFL (SEQ ID NO: 1)-based transportmolecule had the highest uptake (FIG. 5A-C). H/E staining confirmed thatall of the plaques removed were at least AHA Class 5 (fibroatheroma,FIG. 5D). After incubation supernatants were analyzed by gelelectrophoresis. DPRSFL (SEQ ID NO: 1)-based transport molecule was moreefficiently cleaved by human atheroma specimens compared to PLGLAG (SEQID NO: 4)-selective transport molecule, with no cleavage at all of thecontrol mPEG-selective transport molecule (FIG. 5E). Quantitatively,atheromas incubated with DPRSFL (SEQ ID NO: 1)-based transport moleculewere 70% brighter than atheromas incubated in PLGLAG (SEQ ID NO:4)-selective transport molecule, (p=0.03) or mPEG-selective transportmolecule (p=0.002). There was no significant difference between uptakeof PLGLAG (SEQ ID NO: 4)-selective transport molecule and mPEG-selectivetransport molecule (p=0.97, See FIG. S1, Table S3). These resultssuggest that thrombin may be a more sensitive molecular target thanMMP's for targeting advanced human atherosclerotic lesions.

DPRSFL (SEQ ID NO: 1)-Based Transport Molecule can be UsedIntraoperatively for Molecular Navigation in Vascular Surgery.

The utility of DPRSFL (SEQ ID NO: 1) for visualizing potentially lethalvulnerable plaques during fluorescence guided vascular surgery wasassessed by exposing plaque laden arteries in high risk areas such asthe carotid and coronary circulation. Intraoperative (FIGS. 6A and B)and postmortem (FIG. 6C, 6H-I) exposure of the ascending carotid arteryrevealed plaques in the artery itself as well as at the carotidbifurcation. Fluorescence and H/E histology confirmed that plaquecontaining regions of the vessel were bright on fluorescence, whereasplaque-free regions were indistinguishable from background (FIG. 6D-G).We found that DPRSFL (SEQ ID NO: 1)-based transport molecule was moreeffective in labeling carotid plaques than PLGLAG (SEQ ID NO:4)-selective transport molecule (FIG. 6H-J). In one of the oldest,sickest animals, coronary artery plaques were visible with the TRITClabeled DPRSFL (SEQ ID NO: 1)-based transport molecule. Plaques in boththe right and left coronary artery were confirmed by H/E histology (FIG.S6).

Example 3: Thrombin Cleavable Selective Transport Molecules in Cancer,Stroke and Atherosclerosis

Peptide Synthesis.

Peptides (Suc-e9-XDPRSFL-r9-c(cy5)-CONH2,Suc-e9-ODPRSFL-r9-c(cy5)-CONH2, Suc-e9-Xdprsfl-r9-c(cy5)-CONH2 andSuc-e9-(PEG-5)-r9-c(cy5)-CONH2 were synthesized and labeled with cy5using a standard procedure, where X=aminohexanoic acid and O=PEG-2.Briefly, peptides were synthesized on an automatic peptide synthesizerfollowing standard procedures for fluorenylmethoxycarbonyl solid-phasesynthesis. Peptides were N-terminally capped with a succinate andC-termini were amidated. After cleavage off the resin, the C-terminiwere labeled through the cysteine with Cy5 monomaleimide. Peptides werepurified using HPLC.

Cleavage Experiments.

Whole blood was collected either via cardiac puncture or from theabdominal aorta, either in heparinized tubes (plasma) or eppendorf tubes(serum). Both tubes were centrifuged at low speeds to pellet out the redblood cells, and the supernatant was removed and frozen for future use.Assays were done using between 2 and 5 μM peptide. Peptide gels were runand bands were quantified by integrated density using Image J.

Tumor Models

Tumor xenografts were made by inoculating the mammary fatpad withroughly one million cells approximately one week prior to theexperiment. Tumors were allowed to reach no more than 2 cm in size andwere typically in the range of 0.5-1 cm.

Imaging Experiments

Animals were anesthetized with a ketamine/midazolam cocktail (80 mg/kg,40 mg/kg) and injected with the indicated probe (10 nmole/mouse).Animals may have been re-anesthetized at two hours post injection.Animals were sacrificed between four and six hours after injection.Organs were removed and lungs were inflated with 50% OCT/PBS and frozenfor cryohistology. Lung and Tumor imaging.

Twenty micron slices were cut using a cryotome. Images were taken usinga fluorescence dissecting microscope (Zeiss Lumar, ex 620/60, em 700/75)and processed using Adobe Photoshop.

Clot Aging Experiments.

Blood was obtained via cardiac puncture and allowed to clot in anEppendorf tube. Small clots were cut off for processing at each timepoint. At the indicated time point, clots were incubated in either 3 and6 μM Suc-e8-ODPRSFL-c(cy5)-CONH2 or Suc-e8-mPEG-c(cy5)-CONH2 for 10minutes. At least two clots were assayed for each time point for eachmouse. After staining, clots were washed three times in 2 mL PBSshaking, then were imaged at 700 nm using a fluorescence mouse imager(CRI, Maestro, ex 640/48, em 700 nm). Data were quantified byintegration with Image J.

Stroke Experiments.

Male Sprague-Dawley rats (Harlan, San Diego, Calif., USA), 260 to 320 g,underwent surgery to transiently occlude the left middle cerebral artery(tMCAo) with intraluminal 4-0 nylon suture All sutures were pre-bluntedin a microforge (Narishige MF83, NY, USA) and only filaments between 280and 305 μM in diameter were used for occlusion. The surgical exposureand occlusion conditions were performed as previously described. Animalswere prepared with 2 h of tMCAO; rats in the 4 hour reperfusion groupalso received intravenous injections of 2 Mda fluorescein dextran priorto surgery. The rats were re-anesthetized for de-occlusion withisofluorane; immediately following de-occlusion, the tail vein wasinjected with cy5 labeled thrombin probe, cy5 labeled mpeg controlprobe, or cy5 labeled PEG-5 control probe followed by rhodamine labeledthrombin cleavable probe. Reperfusion duration was varied from 4 hours,24 hours, to 48 hours. Rats were then administered an overdose ofpentobarbital and perfused transcardially with normal saline followed bybuffered paraformaldehyde and the brain was removed from the skull.Imaging was done with the Maestro mouse imager (ex 640/48, em 700 or 730nm)

Microstroke Experiments.

Briefly, rats were induced with 4% (v/v) isoflurane and maintained with1 to 2% (v/v) isoflurane in 30% O₂ and 70% N₂O delivered through acustom nose cone. Atropine, 0.05 mg per kg rat, delivered byintraperitoneal injection, and lidocaine, 2% (v/v), delivered bysubcutaneous injection at the site of incision, were administered at thestart of surgery. Body temperature was maintained at 37° C. with afeedback regulated rectal probe and heat pad (50-7053-F; Harvard). Heartrate and blood oxygen saturation were continuously monitored using apulse oximeter (8600V; Nonin). To form the cranial window, the rat wasplaced in a stereotaxic frame and the skull was exposed by a mid-lineincision to the scalp. The left temporal muscle was retracted from theskull and the skull surface was cleaned, dried, and the position of a4×4 mm cranial window, centered at 4.5 mm lateral and −3 mm caudal, wasmarked. Two small anchoring screws (#000 self-tapping; Small Parts) wereplaced rostral and caudal to the window and were glued in place with adrop of Vetbond™. A metal plate for securing the rat to the imagingapparatus was fixed to the skull and screws using dental cement. Thecranial window was generated by first thinning away half of the skullthickness using dental air-drill bit. The perimeter of the window wasfurther thinned until it formed a flexure that revealed the underlyingdura. The bone flap was separated along the flexure using sharp forcepsand gently lifted parallel to the skull surface. The dura was flushedwith ACSF and moist Surgifoam was applied to control any dural vesselbleeding. The dura was then nicked with a 26 gauge needle point andteased apart with two sharp forceps without tearing of any large duralvessels. Low melting-point agarose (#A4018; Sigma), dissolved at aconcentration of 1.5% (w/v) in ACSF containing neither carbonate norphosphate, was cooled to body temperature and applied directly to theexposed cortex. The window was then sealed with a glass cover slip and athin frame was then screwed atop the metal plate to sandwich the coverglass in place. Probe was injected using a tail vein catheter while theanimal was still under anesthesia.

Two photon images were collected using a two-photon laser scanningmicroscope of local design that was controlled by MPScope software. Tomake the vasculature fluorescent, a 0.3 mL bolus of 5% (w/v) 2 MDafluoroscein-dextran (Sigma) in saline was administered through thefemoral artery catheter. A 0.3-numerical aperture (NA), 10-timesmagnification water-dipping objective (Zeiss) was used to collect alarge-scale map to aid navigation through the cortical vasculature. Wethen switched to a 0.8-NA, 40-times magnification water-dippingobjective (Olympus) to obtain high-resolution line-scan and planar imagedata. The line-scans were collected along the centerline of each vesselover a length of 70 pixels spanning 7 to 76 μm, at a scan rate of 1.6kHz/line, to establish the speed of RBCs. Planar image stacks, 256 by256 pixels, were acquired to establish the diameter the vessel. Clotswere generated by injecting rose bengal into the circulation and thenlocally photoactivating it for 1-3 minutes using 532 nm laser light.Several clots were made over the course of 30 minutes prior to injectionof probe. Three hours after probe injection, animals were sacrificed,perfused transcardially with FITC-agarose and the brain removed from theskull. The cortex was flattened and imaged using standard fluorescencemicroscopy (Zeiss).

Atherosclerosis Experiments.

Atherosclerotic animals had a homozygous deletion for ApoE and were feda high fat diet for six to 12 months prior to the experiment. Animalswere injected through the tail vein with probe (10 nmole/mouse). Sixhours later, animals were sacrificed, perfused with saline and aortasharvested. Kidney signal was used to verify that the negative controlanimals received adequate probe.

A Thrombin-Cleavable Peptide was Made by Replacing the XPLGLAG (SEQ IDNO: 18) Linker by (X/O)DPRSFL (SEQ ID NO: 19).

The peptide was labeled with cy5 for detection, purified by HPLC andtested for purity by gel electrophoresis. The peptide was cleaved bypurified thrombin (Calbiochem), with a k_(cat)/k_(m) on the order of5*10⁵, and by purified plasmin (Calbiochem) with a k_(cat)/k_(m) on theorder of 1×10⁵. It was cleaved by serum, but not by heparinized plasma(FIG. 7). Quantitatively, cleavage was detectable by gel when peptidewas incubated by as little as 2.5 nM thrombin. The cleavage by clottedblood was partially inhibited when the assay was done in the presence of200 U/mL hirudin, a natural inhibitor of thrombin derived from leechsaliva. We were not surprised by the incomplete inhibition, sinceincomplete inhibition by direct thrombin inhibitors has been reportedpreviously in the literature for clot-bound thrombin. Another possibleexplanation is activation of Factor Xa, a second enzyme involved in theclotting cascade that is capable of cleaving our probes in vitro(Jessica Crisp, unpublished observation). The one drawback with theXDPRSFL (SEQ ID NO: 20) peptide was that it was not very soluble (up to200 μm in water). Solubility was partially addressed by changing thelinker between the e9 and the r9 from aminohexanoic acid (X) to PEG-2(O). This resulted in little change to the cleavage characteristics ofthe molecule; cleavage by serum was still inhibited by the presence ofheparin and by the addition of 200 U/mL hirudin, but did cause aqualitative improvement in solubility that was necessary for injectioninto animals.

Fluorescent Thrombin Cleavable Probes Accumulate in Tumors inXenografted Animals.

Thrombin and its PAR-1 receptor have been reported to be critical forthe metastatic process in both melanoma and prostate tumor models. Totest whether the thrombin probe could be used to monitor thrombinactivity in vivo, we turned to two of the tumor models used before,HT-1080 xenografts in nude mice (FIG. 8) and B16-F10 grafts intosyngenic immunocompetent B16 albino hosts (FIG. 9). When injectedthrough the tail vein with either cleavable or uncleavable thrombin,tumors were readily visible in both models. Skin background wassignificantly less than for the MMP-based probe, and though synovialuptake remained, there was little uptake in cartilage in either model.The plasma halflife of the peptide was roughly 15 minutes, and excretionwas very clearly via the hepatobiliary system with intestines and liverbecoming very bright by two hours. In both models, uptake measured asper SUV was increased over the PEG-5 control, though overall uptakevaried greatly and in neither case was the average difference betweentest and control significant (Table 6.1). This was explained nicely byfrozen section histology, in which single cells stained extremelybrightly, up to 100-fold over neighboring tissue, both tumor and normal.Though their role is unclear, these cells were uniformly located inareas where tumor was invading muscle. No such staining was seen whenanimals were injected with probes containing the MMP-cleavable linker orthe uncleavable PEG-5 control linker, suggesting that uptake is thrombindependent, although the cause for high uptake in selected cells remainsunclear.

Thrombin Cleavable Probes Accumulate in Tumors, Metastases and Clots inLungs Resulting from Spontaneous Tumors Arising in Transgenic AnimalsExpressing the PyMT Oncogene Driven by the MMTV Promoter

When the thrombin cleavable selective transport molecules were injectedinto tumor bearing PyMT mice, gross tumor fluorescence was notsignificantly different for the cleavable probes relative to the PEG-5control probe. On histology, a dim stromal distribution was punctuatedby bright intratumoral clots and cysts. Additionally, select musclecells appeared to stain if they were infiltrated by or came in directcontact with tumor. This was not the case if the muscle was separatedfrom tumor by a fibrous capsule, indicating that thrombin is likelyimportant in the invasion process but not in the process of tumorestablishment or encapsulation. As expected from previous results,necrotic areas were highlighted with all selective transportmolecule-based probes.

Spontaneous metastases in lung were highlighted by the cleavable probe(n=1), but were nearly invisible for the uncleavable probe (n=1),indicating that the coagulation cascade may be activated by the processof metastasis (FIG. 10). In addition to overt metastases, lungs fromanimals having received the cleavable, but not the uncleavable, peptidecontained very bright clusters of immune cells. The cause for theclusters was not clear, but it is possible that they are due toplatelets and activated immune cells surrounding tumors (FIG. 11). Thethird and most obvious observation made in polyoma lungs was thepresence of very bright clots (FIG. 12). These presented with up to 10:1contrast on gross dissection, and up to 80:1 on histology. The clotswere of unknown etiology, but are probably due to a hypercoaguable stateresulting from high tumor burden and possible metastasis.

Ex-Vivo Clot Aging Studies.

According to the thrombin burst hypothesis, thrombin activity couldtheoretically be used to determine the age of a clot. To test whetherthis could work ex vivo, blood was collected from wildtype mice bycardiac puncture. Clots were allowed to form, and once they had grownlarge enough to be maneuverable were stained for 15 minutes in 5 μMthrombin cleavable XDPRSFL (SEQ ID NO: 20) peptide, washed five timesand imaged. The uncleavable PEG-5 control was used as a standard. Asexpected, the ratio of fluorescence from clots incubated in cleavablepeptide to those incubated in uncleavable peptide started off high, thendecreased steadily over two hours (FIG. 13). The experiment was repeatedmore carefully with blood from three mice with the new ODPRSFL (SEQ IDNO: 21) peptide, yielding similar, but this time statisticallysignificant differences between clots incubated in ODPRSFL (SEQ ID NO:21) peptide and clots incubated in PEG-5 peptide at the 5 m, 20 m and 1h time points. By the two hour time point, there was no significantdifference in uptake between the two peptides. Two clots were done pertime point and the results averaged. The incubation solutions were runon gels after the completion of the experiment, showing incompletecleavage of the ODPRSFL (SEQ ID NO: 21) peptide and no cleavage of thePEG-5 control peptide.

Imaging Stroke with Thrombin Cleavable Selective Transport Molecules

To determine whether thrombin activity could label fresh strokes invivo, we tested our probes in an occlusion/reperfusion model in whichischemic stroke is simulated by temporarily restricting blood flow tothe middle cerebral artery by inserting a microfilament into theinternal carotid artery. Although the blood brain barrier wouldordinarily prevent peptide agents from entering brain parenchyma,ischemia is known to cause blood brain barrier breakdown in theviscinity of the lesion. Upon reperfusion, three animals were given50nmol thrombin-cleavable peptide and three were given the PEG-5 controlpeptide. Animals were sacrificed and perfused 24 hours post occlusion(FIG. 14). Two of the animals given the PEG-5 negative control peptidewere also given a rhodamine labeled thrombin cleavable peptide later ina different color to verify the presence of a clot (FIG. 15).Co-injection of a cy5 labeled thrombin cleavable peptide and a FITClabeled plasmin cleavable peptide gave differential uptake, particularlyin brain parenchyma spatially removed from the damaged vessel (FIG. 16).

In a second preliminary experiment, penetrating arterioles wereindividually clotted by targeted photodynamic damage induced through theexcitation of injected Rose Bengal by a green laser. Excitation of RoseBengal releases singlet oxygen that damages the vessel wall, forming aclot. These clots can either be transient if they are small, or morepermanent if they are larger. Using this strategy, we were able tovisualize clotting of a single arteriole using the thrombin probe withapproximately 2.5 to one contrast in flattened cortex. This strategy forinduction of clots is less traumatic to the animal, and causes lessleakage of the vessels as detected by the FITC-dextran (FIG. 17). Inprinciple, these experiments can also be done in vivo using thetwo-photon microscope. Although preliminary, these results suggest thatthe thrombin peptide may be useful for looking at acute thrombinactivity following ischemic stroke in various models.

Thrombin Activity in Atherosclerosis

A third area where thrombin activity has been correlated to chronicdisease is in the formation of atherosclerotic plaques. Although mostcurrent imaging research on proteases in atherosclerosis focuses on theinvolvement of MMP's and cathepsins PAR's are known to be overexpressedby several cell types in atherosclerosis. Since it was unclear whetherthrombin or MMP would be superior at visualizing plaques, we decided totry both. We collaborated with the Tsimikas lab at UCSD to obtainApoE^(−/−) animals that had been fed a high fat diet and had significant(30-90%) atherosclerotic burden. Since limited animals were available,both male and female end-stage animals were used for this preliminaryset of experiments in which two animals each were injected with MMP,thrombin and the uncleavable mPEG control probe. Animals were quite ill,frequently showing evidence of co-morbidities such as arthritis,blindness and rectal prolapse. Animals were injected with 10 nmol probeand were sacrificed and perfused with saline six hours later. Followingperfusion, aortas were dissected out and pinned down for grossfluorescence analysis.

On gross analysis, there was a statistically significant 2.7-folddifference between plaque uptake in the animals injected with thethrombin cleavable selective transport molecule and control animalsinjected with the uncleavable control probe (1093±437, n=4 vs. 393±179,n=4, p<0.05). Intensities were derived by masking regions of plaque inAmira software and are given in arbitrary units. In non plaque regions,uptake was comparable, 321±108, n=4 vs. 208±124, n=4. When the groupswere separated into male mice and female mice, it became clear thatplaques from the one year old female mice injected with thrombin probetook up twice as much probe as similar plaques from the two two-year-oldmale mice (1440±275, n=2 vs. 746±139, n=2, FIG. 18, FIG. 19). Theopposite trend held true for the four animals injected with the PEG-5negative control peptide (249±179, n=2 vs. 537±275, n=2), likely due tothe presence of necrosis in vulnerable plaques in the aortic archregion. Animals injected with the MMP cleavable selective transportmolecule showed no obvious difference in uptake between the two sets ofanimals, though the aggregate difference between test and control wassignificant (1072±188, n=4 vs. 393±179, n=4, p<0.05).

To verify that the differences in intensities were due to thrombin andMMP cleavage respectively, we used hirudin and an MMP inhibitor cocktail(GM6001 and SB3CT) to inhibit thrombin and MMP activity respectively.Pre-injection with hirudin¹⁵⁹ 30 minutes prior to injection withthrombin cleavable peptide decreased uptake by 75% (1440±275, n=2 vs.354±179, n=2, p=0.06), whereas a cocktail of MMP inhibitors was onlyable to decrease uptake by 50%, FIG. 20. SB3CT delivered alone showed noeffect on uptake (data no shown). Together, this data shows that uptakeis mostly due to protease specific cleavage of the appropriate linker byatherosclerotic plaques.

Visualizing Thrombin Activity in Plaques with TI MRI.

To determine whether MRI was possible using free peptides in principle,and also to look at whether thrombin could be used as a target proteasefor labeling atherosclerotic mice in vivo, we next injected 300 nmol ofthrombin cleavable peptide into a two year old LDLR deficient mouse thathad been fed a high cholesterol diet for 6-12 months. This animal wasimaged pre-injection, immediately post injection and 12 hours thereafterto determine whether sufficient probe could be injected forvisualization using T1-weighted imaging. On MRI, a new structure didappear (FIG. 21), but the animal clearly had difficulty breathingfollowing injection of the probe. Though the breathing difficultycleared up by the next day, the toxicity was so alarming that the freepeptide approach to T1 MRI was abandoned in favor of PAMAM dendrimers.

Background Fluorescence: Implications for Selective Transport MoleculeDevelopment

As expected, there were profound differences in biodistribution for thethrombin probe compared to the MMP probes. Liver and kidney uptakedecreased significantly, with no uptake in cartilage and significantlylower uptake in skin. Though peptide taken from the liver was cleaved,this could easily have occured postmortem, since it was readily apparentduring imaging that the thrombin cleavable peptides are excreted via thehepatobiliary pathway. Synovium was still problematic and was thelargest source of background.

While preferred embodiments of the present invention have been shown anddescribed herein, it will be obvious to those skilled in the art thatsuch embodiments are provided by way of example only. Numerousvariations, changes, and substitutions will now occur to those skilledin the art without departing from the invention. It should be understoodthat various alternatives to the embodiments of the invention describedherein may be employed in practicing the invention. It is intended thatthe following claims define the scope of the invention and that methodsand structures within the scope of these claims and their equivalents becovered thereby.

What is claimed is:
 1. A molecule having the formula: (A-X-B-C)_(n)-Mwherein: C is a fluorescent moiety; A is a peptide with a sequencecomprising a series of 5 glutamate residues; B is a peptide with asequence comprising a series of 8 arginine residues; X is a cleavablelinker comprising PRSFL (SEQ ID NO: 22); M is a macromolecular carrier;and n is an integer between 1 and 20; wherein M is bound to A or B. 2.The molecule of claim 1, wherein X is further attached to6-aminohexanoyl, 5-amino-3-oxapentanoyl, or a combination thereof. 3.The molecule of claim 1, wherein X comprises a sequence selected from:DPRSFL (SEQ ID NO: 1), or PPRSFL (SEQ ID NO: 2).
 4. The molecule ofclaim 1, wherein M is a macromolecular carrier selected from: adendrimer, dextran, a PEG polymer, or albumin.
 5. The molecule of claim1, wherein M is a PEG polymer.
 6. The molecule of claim 1, wherein thefluorescent moiety is an indocarbocyanine dye.
 7. The molecule of claim1, wherein the fluorescent moiety is selected from Cy5, Cy5.5, Cy7,Alexa 647, IRDYE 800CW, or a combination thereof.
 8. The molecule ofclaim 1, wherein the fluorescent moiety is an MRI contrast agent.
 9. Amethod of imaging thrombin activity in a subject, comprising imagingthrombin activity after the subject has been administered a molecule ofthe structure (A-X-B-C)_(n)-M, wherein C is a fluorescent moiety; A is apeptide with a sequence comprising a series of 5 glutamate residues; Bis a peptide with a sequence comprising a series of 8 arginine residues;X is a cleavable linker comprising PRSFL (SEQ ID NO: 22); M is amacromolecular carrier; and n is an integer between 1 and 20; wherein Mis bound to A or B.
 10. The method of claim 9, wherein the fluorescentmoiety is an indocarbocyanine dye.
 11. The method of claim 9, whereinthe fluorescent moiety is selected from Cy5, Cy5.5, Cy7, Alexa 647,IRDYE 800CW, or a combination thereof.